Determining and reducing immunoresistance to a botulinum toxin therapy using botulinum toxin b peptides

ABSTRACT

The present invention provides BoNT/B peptides, BoNT/B peptide compositions, tolerogizing compositions, immune response inducing compositions, as well as methods of determining immunoresistance to botulinum toxin therapy in an individual, methods of treating immunoresistance to botulinum toxin therapy in an individual, methods of reducing anti-botulinum toxin antibodies in an individual and methods of inducing a BoNT/B immune response an individual.

This is a continuation in part that claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 11/755,161, filed May 30, 2007,which claims priority pursuant to 35 U.S.C. §119(e) to U.S. ProvisionalPatent Application Ser. No. 60/810,053 files Jun. 1, 2006, each of whichis hereby incorporated by reference in its entirety.

All patents, patent publications and articles cited in this applicationare hereby incorporated by reference in their entirety.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). Clostridial toxins commerciallyavailable as pharmaceutical compositions include, BoNT/A preparations,such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.),Dysport®/Reloxin®, (Beaufour Ipsen, Porton Down, England), Linurase®(Prollenium, Inc., Ontario, Canada), Neuronox® (Medy-Tox, Inc.,Ochang-myeon, South Korea) BTX-A (Lanzhou Institute Biological Products,China) and Xeomin® (Merz Pharmaceuticals, GmbH., Frankfurt, Germany);and BoNT/B preparations, such as, e.g., MyoBloc™/NeuroBloc™ (ElanPharmaceuticals, San Francisco, Calif.). As an example, BOTOX® iscurrently approved in one or more countries for the followingindications: achalasia, adult spasticity, anal fissure, back pain,blepharospasm, bruxism, cervical dystonia, essential tremor, glabellarlines or hyperkinetic facial lines, headache, hemifacial spasm,hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and VII nerve disorder.

In addition, Clostridial toxin therapies are proposed for treatingneuromuscular disorders, see e.g., Kei Roger Aoki et al., Method forTreating Neuromuscular Disorders and Conditions with Botulinum ToxinTypes A and B, U.S. Pat. No. 6,872,397 (Mar. 29, 2005); Rhett M.Schiffman, Methods for Treating Uterine Disorders, U.S. PatentPublication No. 2004/0175399 (Sep. 9, 2004); Richard L. Barron, Methodsfor Treating Ulcers and Gastroesophageal Reflux Disease, U.S. PatentPublication No. 2004/0086531 (May. 7, 2004); and Kei Roger Aoki, et al.,Method for Treating Dystonia with Botulinum Toxin C to G, U.S. Pat. No.6,319,505 (Nov. 20, 2001); eye disorders, see e.g., Eric R. First,Methods and Compositions for Treating Eye Disorders, U.S. PatentPublication No. 2004/0234532 (Nov. 25, 2004); Kei Roger Aoki et al.,Botulinum Toxin Treatment for Blepharospasm, U.S. Patent Publication No.2004/0151740 (Aug. 5, 2004); and Kei Roger Aoki et al., Botulinum ToxinTreatment for Strabismus, U.S. Patent Publication No. 2004/0126396 (Jul.1, 2004); pain, see e.g., Kei Roger Aoki et al., Pain Treatment byPeripheral Administration of a Neurotoxin, U.S. Pat. No. 6,869,610 (Mar.22, 2005); Stephen Donovan, Clostridial Toxin Derivatives and Methods toTreat Pain, U.S. Pat. No. 6,641,820 (Nov. 4, 2003); Kei Roger Aoki, etal., Method for Treating Pain by Peripheral Administration of aNeurotoxin, U.S. Pat. No. 6,464,986 (Oct. 15, 2002); Kei Roger Aoki andMinglei Cui, Methods for Treating Pain, U.S. Pat. No. 6,113,915 (Sep. 5,2000); Martin A. Voet, Methods for Treating Fibromyalgia, U.S. Pat. No.6,623,742 (Sep. 23, 2003); Martin A. Voet, Botulinum Toxin Therapy forFibromyalgia, U.S. Pat. No. Publication No. 2004/0062776 (Apr. 1, 2004);and Kei Roger Aoki et al., Botulinum Toxin Therapy for Lower Back Pain,U.S. Patent Publication No. 2004/0037852 (Feb. 26, 2004); muscleinjuries, see e.g., Gregory F. Brooks, Methods for Treating MuscleInjuries, U.S. Pat. No. 6,423,319 (Jul. 23, 2002); headache, see e.g.,Martin Voet, Methods for Treating Sinus Headache, U.S. Pat. No.6,838,434 (Jan. 4, 2005); Kei Roger Aoki et al., Methods for TreatingTension Headache, U.S. Pat. No. 6,776,992 (Aug. 17, 2004); and Kei RogerAoki et al., Method for Treating Headache, U.S. Pat. No. 6,458,365 (Oct.1, 2002); William J. Binder, Method for Reduction of Migraine HeadachePain, U.S. Pat. No. 5,714,469 (Feb. 3, 1998); cardiovascular diseases,see e.g., Gregory F. Brooks and Stephen Donovan, Methods for TreatingCardiovascular Diseases with Botulinum Toxin, U.S. Pat. No. 6,767,544(Jul. 27, 2004); neurological disorders, see e.g., Stephen Donovan,Parkinson's Disease Treatment, U.S. Pat. No. 6,620,415 (Sep. 16, 2003);and Stephen Donovan, Method for Treating Parkinson's Disease with aBotulinum Toxin, U.S. Pat. No. 6,306,403 (Oct. 23, 2001);neuropsychiatric disorders, see e.g., Stephen Donovan, Botulinum ToxinTherapy for Neuropsychiatric Disorders, U.S. Patent Publication No.2004/0180061 (Sep. 16, 2004); and Steven Donovan, Therapeutic Treatmentsfor Neuropsychiatric Disorders, U.S. Patent Publication No. 2003/0211121(Nov. 13, 2003); endocrine disorders, see e.g., Stephen Donovan, Methodfor Treating Endocrine Disorders, U.S. Pat. No. 6,827,931 (Dec. 7,2004); Stephen Donovan, Method for Treating Thyroid Disorders with aBotulinum Toxin, U.S. Pat. No. 6,740,321 (May. 25, 2004); Kei Roger Aokiet al., Method for Treating a Cholinergic Influenced Sweat Gland, U.S.Pat. No. 6,683,049 (Jan. 27, 2004); Stephen Donovan, Neurotoxin Therapyfor Diabetes, U.S. Pat. No. 6,416,765 (Jul. 9, 2002); Stephen Donovan,Methods for Treating Diabetes, U.S. Pat. No. 6,337,075 (Jan. 8, 2002);Stephen Donovan, Method for Treating a Pancreatic Disorder with aNeurotoxin, U.S. Pat. No. 6,261,572 (Jul. 17, 2001); Stephen Donovan,Methods for Treating Pancreatic Disorders, U.S. Pat. No. 6,143,306 (Nov.7, 2000); cancers, see e.g., Stephen Donovan, Methods for Treating BoneTumors, U.S. Pat. No. 6,565,870 (May 20, 2003); Stephen Donovan, Methodfor Treating Cancer with a Neurotoxin to Improve Patient Function, U.S.Pat. No. 6,368,605 (Apr. 9, 2002); Stephen Donovan, Method for TreatingCancer with a Neurotoxin, U.S. Pat. No. 6,139,845 (Oct. 31, 2000); andMitchell F. Brin and Stephen Donovan, Methods for Treating DiverseCancers, U.S. Patent Publication No. 2005/0031648 (Feb. 10, 2005); oticdisorders, see e.g., Stephen Donovan, Neurotoxin Therapy for Inner EarDisorders, U.S. Pat. No. 6,358,926 (Mar. 19, 2002); and Stephen Donovan,Method for Treating Otic Disorders, U.S. Pat. No. 6,265,379 (Jul. 24,2001); autonomic disorders, see, e.g., Pankai J. Pasricha and Anthony N.Kalloo, Method for Treating Gastrointestinal Muscle Disorders and OtherSmooth Muscle Dysfunction, U.S. Pat. No. 5,437,291 (Aug. 1, 1995); aswell as other disorders, see e.g., William J. Binder, Method forTreatment of Skin Lesions Associated with Cutaneous Cell-proliferativeDisorders, U.S. Pat. No. 5,670,484 (Sep. 23, 1997); Eric R. First,Application of Botulinum Toxin to the Management of NeurogenicInflammatory Disorders, U.S. Pat. No. 6,063,768 (May 16, 2000); MarvinSchwartz and Brian J. Freund, Method to Reduce Hair Loss and StimulateHair Growth, U.S. Pat. No. 6,299,893 (Oct. 9, 2001); Jean D. A.Carruthers and Alastair Carruthers, Cosmetic Use of Botulinum Toxin forTreatment of Downturned Mouth, U.S. Pat. No. 6,358,917 (Mar. 19, 2002);Stephen Donovan, Use of a Clostridial Toxin to Reduce Appetite, U.S.Patent Publication No. 2004/40253274 (Dec. 16, 2004); and Howard I. Katzand Andrew M. Blumenfeld, Botulinum Toxin Dental Therapies andProcedures, U.S. Patent Publication No. 2004/0115139 (Jun. 17, 2004);Kei Roger Aoki, et al., Treatment of Neuromuscular Disorders andConditions with Different Botulinum, U.S. Patent Publication No.2002/0010138 (Jan. 24, 2002); and Kei Roger Aoki, et al., Use ofBotulinum Toxins for Treating Various Disorders and Conditions andAssociated Pain, U.S. Patent Publication No. 2004/0013692 (Jan. 22,2004). In addition, the expected use of Clostridial toxins, such as,e.g., BoNTs and TeNT, in therapeutic and cosmetic treatments of humansand other mammals is anticipated to expand to an ever widening range ofdiseases and ailments that can benefit from the properties of thesetoxins.

While a potent and effective treatment, the inhibition ofneurotransmitter release and the resulting neuromuscular paralysiselicited by Clostridial toxin therapies is not permanent. The reversiblenature of these paralytic effects requires periodic treatments in orderto maintain the therapeutic benefits from this toxin. As a consequenceof this repeated exposure, an immune response against a Clostridialtoxin can occur in some patients which reduce or completely prevent theindividual's responsiveness to further treatments, see, e.g., JosephJankovic, Botulinum toxin: Clinical Implications of Antigenicity andImmunoresistance, (SCIENTIFIC AND THERAPEUTIC ASPECTS OF BOTULINUMTOXIN, 409-415, Mitchell F. Brin et al., eds., Lippincott Williams &Wilkins, 2002); Dirk Dressler, Clinical Presentation and Management ofAntibody-induced Failure of Botulinum Toxin Therapy, 19(Suppl. 8) MOV.DISORD. S92-S100 (2004); M. Zouhair Atassi, Basic Immunological Aspectsof Botulinum Toxin Therapy, 19(Suppl. 8) MOV. DISORD. S68-S84, (2004).

Thus, there exists a need for methods of determining immunoresistance inan individual to Clostridial toxin therapy, methods of preventing orreducing immunoresistance in an individual to Clostridial toxin therapyas well as compositions to carry out these methods. The presentinvention satisfies this need with respect to BoNT/B therapies andprovides additional related advantages as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of human anti-pentavalent botulinum toxoidantibodies to overlapping synthetic BoNT/B peptides spanning the BoNT/BH_(N) and H_(C) domains of BoNT/B. Also shown is the binding tofull-length BoNT/B as a positive control.

FIG. 2 shows binding of horse anti-BoNT/B antibodies to overlappingsynthetic BoNT/B peptides spanning the BoNT/B H_(N) and H_(C) domains ofBoNT/B. Also shown is the binding to full-length BoNT/B as a positivecontrol.

FIG. 3 shows binding of anti-BoNT/B antibodies of ICR outbred mice tooverlapping synthetic BoNT/B peptides spanning the BoNT/B H_(N) andH_(C) domains of BoNT/B. Also shown is the binding to full-length BoNT/Bas a positive controls.

FIG. 4 shows a comparison of the BoNT/B peptide-binding profiles ofhuman, horse and mouse antibodies against BoNT/B. The data for horse andmouse antisera are shown here at dilutions of 1:500, (vol/vol), whilehuman antisera are at dilution of 1:1000 (vol/vol) in PBS/0.1% BSA.

FIG. 5 shows the BoNT/B peptide-binding profiles against sera fromMPA-positive CD patients. FIG. 5 a shows the binding profiles ofanti-BoNT/B antiserum to H-chain peptides of BoNT/B. The anti-BoNT/Bantiserum was a mixture of equal volumes of antisera from 30BoNT/B-treated CD patients. The assays were done at antisera dilutionsof 1:500 (vol/vol). The results are expressed in ratio of cpm bound topeptide/cpm bound to controls of unrelated protein (BSA) or peptides.FIG. 5 b shows the recognition frequency of each H-chain peptide inpercent of the 30 sera. The darkest bars represent peptides that arerecognized by over 15 out of 30 (50%) of the sera. Lighter bars indicaterecognition by 12 to 9 sera and white bars by 6 or less sera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the discovery of BoNT/B peptides whichelicit antibody responses and represent the complete repertoire ofepitopes found within the H_(N) domain and H_(C) domain of the BoNT/Bheavy chain recognized by anti-BoNT/B antibodies present in three animalspecies, including humans. As shown herein in Examples 1-5, antigenicregions of both domains were mapped using human, horse and mouse seraobtained following immunization with BoNT/B. Mapping was performed usingtwenty-nine synthetic BoNT/B peptides, each containing nineteenresidues, that overlap consecutively by five residues and correspond tothe entire length of the H_(N) domain (Table 2) and thirty-one syntheticBoNT/B peptides, each containing nineteen residues, that overlapconsecutively by five residues and correspond to the entire length ofthe H_(C) domain, with the exception of C31, which is twenty-tworesidues in length (Table 3). Results from the mapping studiesrevealed 1) four segments of BoNT/B that represent the completerepertoire of continuous antigenic regions on the BoNT/B H_(N) domain;and 2) 1) eleven segments of BoNT/B that represent the completerepertoire of continuous antigenic regions on the BoNT/B H_(C) domain,see, e.g., Examples 1-5. BoNT/B peptides of the present invention areuseful for, e.g., making peptides and peptide compositions and employingmethods for determining immunoresistance to a botulinum toxin therapy inan individual, making tolerogizing compositions and employing methodsfor treating immunoresistance to a botulinum toxin therapy in anindividual, making an immune response inducing composition and employingmethods of inducing an immune response in an individual and methods ofproducing an anti-BoNT/B antibody.

Thus, aspects of the present invention provide BoNT/B peptides having alength of at least 5 amino acids and at most 60 amino acids. In otheraspects of this embodiment, such a BoNT/B peptide is derived from anaturally occurring BoNT/B, such as, e.g., a BoNT/B isoform or a BoNT/Bsubtype. In still other aspects of this embodiment, such a BoNT/Bpeptide is derived from a non-naturally occurring BoNT/B, such as, e.g.,a conservative BoNT/B variant, a non-conservative BoNT/B variant, or achimeric BoNT/B variant. In yet another aspect of this embodiment, sucha BoNT/B peptide comprises an immunoreactive fragment of the BoNT/Bpeptide, the BoNT/B peptide derived from a naturally occurring BoNT/B ora non-naturally occurring BoNT/B.

In other aspects of this embodiment, a BoNT/B peptide comprises a BoNT/Bpeptide of SEQ ID NO: 1 having a length of at least 5 amino acids and atmost 60 amino acids, a BoNT/B peptide of SEQ ID NO: 3 having a length ofat least 5 amino acids and at most 60 amino acids, a BoNT/B peptide ofSEQ ID NO: 5 having a length of at least 5 amino acids and at most 60amino acids, a BoNT/B peptide of SEQ ID NO: 7 having a length of atleast 5 amino acids and at most 60 amino acids, a BoNT/B peptide of SEQID NO: 9 having a length of at least 5 amino acids and at most 60 aminoacids, a BoNT/B peptide of SEQ ID NO: 11 having a length of at least 5amino acids and at most 60 amino acids or a BoNT/B peptide of SEQ ID NO:11 having a length of at least 5 amino acids and at most 60 amino acids.

In still other aspects of this embodiment, such a BoNT/B peptidecomprises an amino acid sequence selected from the group consisting ofamino acids 610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO:1, amino acids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ IDNO: 1, amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQID NO: 1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838 ofSEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880of SEQ ID NO: 1, amino acids 862-880 of SEQ ID NO: 7, amino acids890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3, aminoacids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 7,amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ ID NO:3, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ IDNO: 5, amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQID NO: 3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992 ofSEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, aminoacids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1,amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ IDNO: 5, amino acids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 ofSEQ ID NO: 1, amino acids 1072-1090 of SEQ ID NO: 3, amino acids1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1, aminoacids 1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1,amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, such a BoNT/B peptidecomprises an amino acid sequence selected from the group consisting ofamino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO:5, amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ IDNO: 5, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 ofSEQ ID NO: 7, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950of SEQ ID NO: 5, amino acids 974-992 of SEQ ID NO: 1, amino acids974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, aminoacids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3,amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ IDNO: 7, amino acids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 ofSEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5 and amino acids1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, such a BoNT/B peptidecomprises an amino acid sequence selected from the group consisting ofamino acids 616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO:1, amino acids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ IDNO: 1, amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQID NO: 7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 ofSEQ ID NO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984of SEQ ID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids974-984 of SEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, aminoacids 1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7,amino acids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ IDNO: 3, amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 ofSEQ ID NO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 oramino acids 1269-1281 of SEQ ID NO: 7.

Other aspects of the present invention provide BoNT/B peptidecompositions comprising a BoNT/B peptide having a length of at least 5amino acids and at most 60 amino acids. It is envisioned that any andall BoNT/B peptides disclosed in the present specification that producea decreased immunological response can be useful in such a tolerogizingcomposition, including, without limitation, a BoNT/B derived from anaturally occurring BoNT/B, a BoNT/B derived from a non-naturallyoccurring BoNT/B and a BoNT/B comprising an immunoreactive fragment ofthe BoNT/B peptide, the BoNT/B peptide derived from a naturallyoccurring BoNT/B or a non-naturally occurring BoNT/B.

Other aspects of the present invention provide tolerogizing compositionscomprising a tolerogizing agent and a BoNT/B peptide. It is envisionedthat any and all tolerogizing agents can be useful in such atolerogizing composition, including, without limitation, polyethyleneglycol (PEG), monomethoxypolyethylene glycol (mPEG) and polyvinylalcohol (PVA). It is also envisioned that any and all BoNT/B peptidesdisclosed in the present specification that produce a decreasedimmunological response can be useful in such a tolerogizing composition,including, without limitation, a BoNT/B derived from a naturallyoccurring BoNT/B, a BoNT/B derived from a non-naturally occurring BoNT/Band a BoNT/B comprising an immunoreactive fragment of the BoNT/Bpeptide, the BoNT/B peptide derived from a naturally occurring BoNT/B ora non-naturally occurring BoNT/B.

Other aspects of the present invention provide immune response inducingcompositions comprising an adjuvant and a BoNT/B antigen. It isenvisioned that any and all adjuvants can be useful in such an immuneresponse inducing composition, including, without limitation,polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG) andpolyvinyl alcohol (PVA). It is also envisioned that any and all BoNT/Bpeptides disclosed in the present specification that produce animmunological response can be useful as a BoNT/B antigen, including,without limitation, a BoNT/B derived from a naturally occurring BoNT/B,a BoNT/B derived from a non-naturally occurring BoNT/B and a BoNT/Bcomprising an immunoreactive fragment of the BoNT/B peptide, the BoNT/Bpeptide derived from a naturally occurring BoNT/B or a non-naturallyoccurring BoNT/B.

Still other aspects of the present invention provide methods ofdetermining immunoresistance to BoNT therapy in an individual, themethods comprising the steps of combining a BoNT/B peptide and a testsample under conditions suitable for the selective binding of the BoNT/Bpeptide to an anti-BoNT antibody and determining the presence of ananti-BoNT antibody-BoNT/B peptide complex, the antibody-peptide complexformed by the selective binding of an anti-BoNT antibody and the BoNT/Bpeptide, where the presence of the anti-BoNT antibody-BoNT/B peptidecomplex is indicative of immunoresistance to a BoNT therapy. It isenvisioned that any and all BoNT/B peptides disclosed in the presentspecification can be useful in a method for determining immunoresistanceto botulinum toxin therapy in an individual, including, withoutlimitation, a BoNT/B derived from a naturally occurring BoNT/B, a BoNT/Bderived from a non-naturally occurring BoNT/B and a BoNT/B comprising animmunoreactive fragment of the BoNT/B peptide, the BoNT/B peptidederived from a naturally occurring BoNT/B or a non-naturally occurringBoNT/B.

Still other aspects of the present invention provide methods of treatingimmunoresistance to botulinum toxin therapy in an individual, the methodcomprising the step of administering to the individual a tolerogizingcomposition, the tolerogizing composition comprising a tolerogizingagent conjugated to a BoNT/B peptide, where the administration of thetolerogizing composition producing a decrease in an immunologicalresponse against the botulinum toxin therapy in the individual. It isenvisioned that any and all BoNT/B peptides disclosed in the presentspecification can be useful in a method from preventing or reducingimmunoresistance to botulinum toxin therapy in an individual, including,without limitation, a BoNT/B derived from a naturally occurring BoNT/B,a BoNT/B derived from a non-naturally occurring BoNT/B and a BoNT/Bcomprising an immunoreactive fragment of the BoNT/B peptide, the BoNT/Bpeptide derived from a naturally occurring BoNT/B or a non-naturallyoccurring BoNT/B.

Still other aspects of the present invention provide anti-BoNTimmunoapheresis methods of treating immunoresistance to a BoNT therapyin an individual, the method comprising the steps of contacting ananti-BoNT antibody containing component from the individualextracorporeally with a BoNT/B peptide immunosorbent under conditionssuitable for the selective binding of the BoNT/B peptide to theanti-BoNT antibody, the BoNT/B peptide and returning the anti-BoNTantibody-depleted component back into the individual. It is envisionedthat any and all BoNT/B peptides disclosed in the present specificationcan be useful in an anti-BoNT immunoapheresis methods of treatingimmunoresistance to a BoNT therapy in an individual, including, withoutlimitation, a BoNT/B derived from a naturally occurring BoNT/B, a BoNT/Bderived from a non-naturally occurring BoNT/B and a BoNT/B comprising animmunoreactive fragment of the BoNT/B peptide, the BoNT/B peptidederived from a naturally occurring BoNT/B or a non-naturally occurringBoNT/B.

Still other aspects of the present invention provide methods of inducinga BoNT/B immune response in an individual, the method comprising thestep of administering to the individual an immune response inducingcomposition comprising an adjuvant and a BoNT/B antigen, whereadministration of the immune response inducing composition produces animmune response in the individual. It is envisioned that any and allBoNT/B peptides disclosed in the present specification can be useful asBoNT/B antigens in methods for inducing an immune response in anindividual, including, without limitation, a BoNT/B derived from anaturally occurring BoNT/B, a BoNT/B derived from a non-naturallyoccurring BoNT/B and a BoNT/B comprising an immunoreactive fragment ofthe BoNT/B peptide, the BoNT/B peptide derived from a naturallyoccurring BoNT/B or a non-naturally occurring BoNT/B.

Yet other aspects of the present invention provide methods of producingan anti-BoNT/B antibody in an individual, the method comprising thesteps of administering to the individual an immune response inducingcomposition comprising an adjuvant and a BoNT/B antigen, whereadministration of the immune response inducing composition produces animmune response in the individual; collecting from the individual asample containing the anti-BoNT/B antibody or anti-BoNT/Bantibody-producing cell; and isolating the anti-BoNT/B antibody from thesample. It is envisioned that antibodies produced according to a methodof the invention include polyclonal and monoclonal antibodies. It isalso envisioned that any and all BoNT/B peptides disclosed in thepresent specification can be useful as BoNT/B antigens in methods ofproducing an anti-BoNT/B antibody in an individual, including, withoutlimitation, a BoNT/B derived from a naturally occurring BoNT/B, a BoNT/Bderived from a non-naturally occurring BoNT/B and a BoNT/B comprising animmunoreactive fragment of the BoNT/B peptide, the BoNT/B peptidederived from a naturally occurring BoNT/B or a non-naturally occurringBoNT/B.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). While all seven botulinum toxins(BoNT) serotypes have similar structure and pharmacological properties,each also displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other species of Clostridia, C. baratii and C. butyricum,also produce toxins similar to BoNT/F and BoNT/E, respectively.

Clostridia toxins possess approximately 35% amino acid identity witheach other and share the same functional domain organization and overallstructural architecture. Clostridial toxins are each translated as asingle chain polypeptide of approximately 150 kDa that is subsequentlycleaved by proteolytic scission within a disulfide loop by anaturally-occurring protease, such as, e.g., an endogenous Clostridialtoxin protease or a naturally-occurring protease produced in theenvironment (FIG. 2). This posttranslational processing yields adi-chain molecule comprising an approximately 50 kDa light chain (LC)and an approximately 100 kDa heavy chain (HC) held together by a singledisulfide bond and noncovalent interactions. Each mature di-chainmolecule comprises three functionally distinct domains: 1) an enzymaticdomain located in the LC that includes a metalloprotease regioncontaining a zinc-dependent endopeptidase activity which specificallytargets core components of the neurotransmitter release apparatus (Table1); 2) a translocation domain contained within the amino-terminal halfof the HC (H_(N)) that facilitates release of the LC from intracellularvesicles into the cytoplasm of the target cell (Table 1); and 3) abinding domain found within the carboxyl-terminal half of the HC (H_(C))that determines the binding activity and binding specificity of thetoxin to the receptor complex located at the surface of the target cell(Table 1).

TABLE 1 Clostridial Toxin Reference Sequences and Regions Toxin SEQ IDNO: LC H_(N) H_(C) BoNT/A 1 M1-K448 A449-K871 N872-L1296 BoNT/B 2M1-K441 A442-S858 E859-E1291 BoNT/C1 3 M1-K449 T450-N866 N867-E1291BoNT/D 4 M1-R445 D446-N862 S863-E1276 BoNT/E 5 M1-R422 K423-K845R846-K1252 BoNT/F 6 M1-K439 A440-K864 K865-E1274 BoNT/G 7 M1-K446S447-S863 N864-E1297 TeNT 8 M1-A457 S458-V879 I880-D1315

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Although the applicants have no wish to be limited by thefollowing description, the intoxication mechanism can be described ascomprising at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) enzymatic targetmodification. The process is initiated when the H_(C) domain of aClostridial toxin binds to a toxin-specific receptor system located onthe plasma membrane surface of a target cell. The binding specificity ofa receptor complex is thought to be achieved, in part, by specificcombinations of gangliosides and protein receptors that appear todistinctly comprise each Clostridial toxin receptor complex. Once bound,the toxin/receptor complexes are internalized by endocytosis and theinternalized vesicles are sorted to specific intracellular routes. Thetranslocation step appears to be triggered by the acidification of thevesicle compartment. This process seems to initiate two importantpH-dependent structural rearrangements that increase hydrophobicity andpromote formation di-chain form of the toxin. Once activated, lightchain endopeptidase of the toxin is released from the intracellularvesicle into the cytosol where it appears to specifically target one ofthree known core components of the neurotransmitter release apparatus.These core proteins, vesicle-associated membrane protein(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa(SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking andfusion at the nerve terminal and constitute members of the solubleN-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE)family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminalregion, releasing a nine or twenty-six amino acid segment, respectively,and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. Thebotulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanustoxin, act on the conserved central portion of VAMP, and release theamino-terminal portion of VAMP into the cytosol. BoNT/C1 cleavessyntaxin at a single site near the cytosolic membrane surface. Theselective proteolysis of synaptic SNAREs accounts for the block ofneurotransmitter release caused by Clostridial toxins in vivo. The SNAREprotein targets of Clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli etal., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11 (9)Trends Microbiol. 431-437, (2003).

The present invention provides, in part, a BoNT/B peptide. As usedherein, the term “BoNT/B peptide,” means a peptide having an amino acidsequence length of at least five amino acids and at most 60 amino acids,the amino acid sequence derived from a naturally occurring BoNT/B or anon-naturally occurring BoNT/B. An exemplary BoNT/B is the BoNT/B of SEQID NO: 1, encoded by the polynucleotide molecule of SEQ ID NO: 2.

In is envisioned that a BoNT/B peptide disclosed in the presentspecification can have any of a variety of lengths from at least 5 aminoacids to at most 60 amino acids. Therefore, aspects of this embodimentmay include a BoNT/B peptide with, e.g., at least five amino acids, atleast six amino acids, at least seven amino acids, at least eight aminoacids, at least nine amino acids, at least ten amino acids, at least 11amino acids, at least 12 amino acids, at least 13 amino acids, at least14 amino acids, at least 15 amino acids, at least 16 amino acids, atleast 17 amino acids, at least 18 amino acids, at least 19 amino acids,at least 20 amino acids, at least 25 amino acids, at least 30 aminoacids, at least 35 amino acids, at least 40 amino acids, at least 45amino acids, at least 50 amino acids, or at least 55 amino acids. Inother aspects of this embodiment may include a BoNT/B peptide with,e.g., at most six amino acids, at most seven amino acids, at most eightamino acids, at most nine amino acids, at most ten amino acids, at most11 amino acids, at most 12 amino acids, at most 13 amino acids, at most14 amino acids, at most 15 amino acids, at most 16 amino acids, at most17 amino acids, at most 18 amino acids, at most 19 amino acids, at most20 amino acids, at most 25 amino acids, at most 30 amino acids, at most35 amino acids, at most 40 amino acids, at most 45 amino acids, at most50 amino acids, at most 55 amino acids or at most 60 amino acids.

A BoNT/B peptide disclosed in the present specification comprises atleast five consecutive amino acids from the BoNT/B from which the BoNT/Bpeptide is derived. In aspects of this embodiment, a BoNT/B peptidecomprises, e.g., at least 5 consecutive amino acids from the BoNT/B fromwhich the BoNT/B peptide is derived, at least 6 consecutive amino acidsfrom the BoNT/B from which the BoNT/B peptide is derived, at least 7consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived, at least 8 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived, at least 9 consecutive amino acids fromthe BoNT/B from which the BoNT/B peptide is derived, at least 10consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived, at least 12 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived, at least 15 consecutive amino acids fromthe BoNT/B from which the BoNT/B peptide is derived, at least 18consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived, at least 20 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived, at least 25 consecutive amino acids fromthe BoNT/B from which the BoNT/B peptide is derived, at least 30consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived, at least 40 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived or at least 50 consecutive amino acidsfrom the BoNT/B from which the BoNT/B peptide is derived.

In other aspects of this embodiment, a BoNT/B peptide comprises, e.g.,at most 5 consecutive amino acids from the BoNT/B from which the BoNT/Bpeptide is derived, at most 6 consecutive amino acids from the BoNT/Bfrom which the BoNT/B peptide is derived, at most 7 consecutive aminoacids from the BoNT/B from which the BoNT/B peptide is derived, at most8 consecutive amino acids from the BoNT/B from which the BoNT/B peptideis derived, at most 9 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived, at most 10 consecutive amino acids fromthe BoNT/B from which the BoNT/B peptide is derived, at most 12consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived, at most 15 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived, at most 18 consecutive amino acids fromthe BoNT/B from which the BoNT/B peptide is derived, at most 20consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived, at most 25 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived, at most 30 consecutive amino acids fromthe BoNT/B from which the BoNT/B peptide is derived, at most 40consecutive amino acids from the BoNT/B from which the BoNT/B peptide isderived or at most 50 consecutive amino acids from the BoNT/B from whichthe BoNT/B peptide is derived.

In still other aspects of this embodiment, a BoNT/B peptide comprising,e.g., at least five amino acids of SEQ ID NO:1, at least six amino acidsof SEQ ID NO:1, at least seven amino acids of SEQ ID NO:1, at leasteight amino acids of SEQ ID NO:1, at least nine amino acids of SEQ IDNO:1, at least ten amino acids of SEQ ID NO:1, at least 11 amino acidsof SEQ ID NO:1, at least 12 amino acids of SEQ ID NO:1, at least 13amino acids of SEQ ID NO:1, at least 14 amino acids of SEQ ID NO:1, atleast 15 amino acids of SEQ ID NO:1, at least 16 amino acids of SEQ IDNO:1, at least 17 amino acids of SEQ ID NO:1, at least 18 amino acids ofSEQ ID NO:1, at least 19 amino acids of SEQ ID NO:1, at least 20 aminoacids of SEQ ID NO:1, at least 25 amino acids of SEQ ID NO:1, at least30 amino acids of SEQ ID NO:1, at least 35 amino acids of SEQ ID NO:1,at least 40 amino acids of SEQ ID NO:1, at least 45 amino acids of SEQID NO:1, at least 50 amino acids of SEQ ID NO:1 or at least 55 aminoacids.

In still other aspects of this embodiment, a BoNT/B peptide with, e.g.,at most six amino acids of SEQ ID NO:1, at most seven amino acids of SEQID NO:1, at most eight amino acids of SEQ ID NO:1, at most nine aminoacids of SEQ ID NO:1, at most ten amino acids of SEQ ID NO:1, at most 11amino acids of SEQ ID NO:1, at most 12 amino acids of SEQ ID NO:1, atmost 13 amino acids of SEQ ID NO:1, at most 14 amino acids of SEQ IDNO:1, at most 15 amino acids of SEQ ID NO:1, at most 16 amino acids ofSEQ ID NO:1, at most 17 amino acids of SEQ ID NO:1, at most 18 aminoacids of SEQ ID NO:1, at most 19 amino acids of SEQ ID NO:1, at most 20amino acids of SEQ ID NO:1, at most 25 amino acids of SEQ ID NO:1, atmost 30 amino acids of SEQ ID NO:1, at most 35 amino acids of SEQ IDNO:1, at most 40 amino acids of SEQ ID NO:1, at most 45 amino acids ofSEQ ID NO:1, at most 50 amino acids of SEQ ID NO:1, at most 55 aminoacids or at most 60 amino acids of SEQ ID NO:1.

In yet other aspects of this embodiment, a BoNT/B peptide comprising,e.g., at least five consecutive amino acids of SEQ ID NO:1, at least sixconsecutive amino acids of SEQ ID NO:1, at least seven consecutive aminoacids of SEQ ID NO:1, at least eight consecutive amino acids of SEQ IDNO:1, at least nine consecutive amino acids of SEQ ID NO:1, at least tenamino consecutive acids of SEQ ID NO:1, at least 11 consecutive aminoacids of SEQ ID NO:1, at least 12 consecutive amino acids of SEQ IDNO:1, at least 13 consecutive amino acids of SEQ ID NO:1, at least 14consecutive amino acids of SEQ ID NO:1, at least 15 consecutive aminoacids of SEQ ID NO:1, at least 16 consecutive amino acids of SEQ IDNO:1, at least 17 consecutive amino acids of SEQ ID NO:1, at least 18consecutive amino acids of SEQ ID NO:1, at least 19 consecutive aminoacids of SEQ ID NO:1, at least 20 consecutive amino acids of SEQ IDNO:1, at least 25 consecutive amino acids of SEQ ID NO:1, at least 30consecutive amino acids of SEQ ID NO:1, at least 35 consecutive aminoacids of SEQ ID NO:1, at least 40 consecutive amino acids of SEQ IDNO:1, at least 45 consecutive amino acids of SEQ ID NO:1, at least 50consecutive amino acids of SEQ ID NO:1 or at least 55 consecutive aminoacids.

In yet other aspects of this embodiment, a BoNT/B peptide with, e.g., atmost five consecutive amino acids of SEQ ID NO:1, at most sixconsecutive amino acids of SEQ ID NO:1, at most seven consecutive aminoacids of SEQ ID NO:1, at most eight consecutive amino acids of SEQ IDNO:1, at most nine consecutive amino acids of SEQ ID NO:1, at most tenconsecutive amino acids of SEQ ID NO:1, at most 11 consecutive aminoacids of SEQ ID NO:1, at most 12 consecutive amino acids of SEQ ID NO:1,at most 13 consecutive amino acids of SEQ ID NO:1, at most 14consecutive amino acids of SEQ ID NO:1, at most 15 consecutive aminoacids of SEQ ID NO:1, at most 16 consecutive amino acids of SEQ ID NO:1,at most 17 consecutive amino acids of SEQ ID NO:1, at most 18consecutive amino acids of SEQ ID NO:1, at most 19 consecutive aminoacids of SEQ ID NO:1, at most 20 consecutive amino acids of SEQ ID NO:1,at most 25 consecutive amino acids of SEQ ID NO:1, at most 30consecutive amino acids of SEQ ID NO:1, at most 35 consecutive aminoacids of SEQ ID NO:1, at most 40 consecutive amino acids of SEQ ID NO:1,at most 45 consecutive amino acids of SEQ ID NO:1, at most 50consecutive amino acids of SEQ ID NO:1, at most 55 consecutive aminoacids or at most 60 consecutive amino acids of SEQ ID NO:1.

A BoNT/B peptide includes, without limitation, a BoNT/B peptidecomprising an amino acid sequence derived from a naturally occurringBoNT/B variant, such as, e.g., a BoNT/B isoform and a BoNT/B subtype;and a BoNT/B peptide comprising an amino acid sequence derived from anon-naturally occurring BoNT/B variant, such as, e.g., a conservativeBoNT/B variant, a non-conservative BoNT/B variant or a chimeric BoNT/Bpeptide.

As used herein, the term “BoNT/B variant,” whether naturally-occurringor non-naturally-occurring, means a BoNT/B that has at least one aminoacid change from the corresponding region of the reference BoNT/B of SEQID NO: 1 and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allBoNT/B variants disclosed in the present specification can function insubstantially the same manner as the reference BoNT/B of SEQ ID NO: 1 onwhich the BoNT/B variant is based, and can be substituted for thereference BoNT/B of SEQ ID NO: 1 in any aspect of the present invention.As a non limiting example, a BoNT/B peptide comprising amino acidsequence 974-992 from a BoNT/B variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid sequence 974-992 of SEQ IDNO: 1. As another non limiting example, a BoNT/B peptide comprisingamino acid sequence 736-754 from a BoNT/B variant will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid sequence 736-754 ofSEQ ID NO: 1. As still another non limiting example, a BoNT/B peptidecomprising amino acid sequence 1058-1076 from a BoNT/B variant will haveat least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acidsequence 1058-1076 of SEQ ID NO: 1. As yet another non limiting example,a BoNT/B peptide comprising amino acid sequence 890-908 from a BoNT/Bvariant will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid sequence 890-908 of SEQ ID NO: 1.

Any of a variety of sequence alignment methods can be used to determinepercent identity of a naturally-occurring BoNT/B variant or anon-naturally-occurring BoNT/B variant, including, without limitation,global methods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics: 1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

It is recognized by those of skill in the art that there are naturallyoccurring BoNT/B variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently four BoNT/B subtypes, BoNT/B1, BoNT/B2,BoNT/B bivalent and BoNT/B non-proteolytic, with specific subtypesshowing approximately 95% amino acid identity when compared to anotherBoNT/B subtype. As used herein, the term “naturally occurring BoNT/Bvariant” means any BoNT/B produced by a naturally-occurring process,including, without limitation, BoNT/B isoforms produced fromalternatively-spliced transcripts, BoNT/B isoforms produced byspontaneous mutation and BoNT/B subtypes. A naturally occurring BoNT/Bvariant can function in substantially the same manner as the referenceBoNT/B of SEQ ID NO: 1 on which the naturally occurring BoNT/B variantis based, and can be substituted for the reference BoNT/B of SEQ ID NO:1 in any aspect of the present invention. A naturally occurring BoNT/Bvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids, ten or more amino acids, 20 or more amino acids, 30 or more aminoacids, 40 or more amino acids, 50 or more amino acids or 100 or moreamino acids from the reference BoNT/B of SEQ ID NO: 1 on which thenaturally occurring BoNT/B variant is based. A naturally occurringBoNT/B variant can also substitute at least 10 contiguous amino acids,at least 15 contiguous amino acids, at least 20 contiguous amino acids,or at least 25 contiguous amino acids from the reference BoNT/B of SEQID NO: 1 on which the naturally occurring BoNT/B variant is based, thatpossess at least 50% amino acid identity, 65% amino acid identity, 75%amino acid identity, 85% amino acid identity or 95% amino acid identityto the reference BoNT/B of SEQ ID NO: 1 on which the naturally occurringBoNT/B variant is based. A non-limiting example of a naturally occurringBoNT/B variant is a BoNT/B isoform.

Another non-limiting example of a naturally occurring BoNT/B variant isa BoNT/B subtype, such as, e.g., BoNT/B1, BoNT/B2, BoNT/Bnon-proteolytic and BoNT/B bivalents. An exemplary BoNT/B1 is the BoNT/Bof SEQ ID NO: 1, encoded by the polynucleotide molecule of SEQ ID NO: 2.An exemplary BoNT/B2 is the BoNT/B of SEQ ID NO: 3, encoded by thepolynucleotide molecule of SEQ ID NO: 4. An exemplary BoNT/Bnon-proteolytic is the BoNT/B of SEQ ID NO: 5, encoded by thepolynucleotide molecule of SEQ ID NO: 6. Exemplary BoNT/B bivalents arethe BoNT/B of SEQ ID NO: 7, encoded by the polynucleotide molecule ofSEQ ID NO: 8; the BoNT/B of SEQ ID NO: 9, encoded by the polynucleotidemolecule of SEQ ID NO: 10; the BoNT/B of SEQ ID NO: 11, encoded by thepolynucleotide molecule of SEQ ID NO: 12; and the BoNT/B of SEQ ID NO:13, encoded by the polynucleotide molecule of SEQ ID NO: 14.

Thus in aspects of this embodiment, a BoNT/B peptide is derived from theBoNT/B of SEQ ID NO: 1, the BoNT/B of SEQ ID NO: 3, the BoNT/B of SEQ IDNO: 5, the BoNT/B of SEQ ID NO: 7, the BoNT/B of SEQ ID NO: 9, theBoNT/B of SEQ ID NO: 11 or the BoNT/B of SEQ ID NO: 13. In other aspectsof this embodiment, a BoNT/B peptide comprises a BoNT/B peptide of SEQID NO: 1 having a length of at least 5 amino acids and at most 60 aminoacids, a BoNT/B peptide of SEQ ID NO: 3 having a length of at least 5amino acids and at most 60 amino acids, a BoNT/B peptide of SEQ ID NO: 5having a length of at least 5 amino acids and at most 60 amino acids, aBoNT/B peptide of SEQ ID NO: 7 having a length of at least 5 amino acidsand at most 60 amino acids, a BoNT/B peptide of SEQ ID NO: 9 having alength of at least 5 amino acids and at most 60 amino acids, a BoNT/Bpeptide of SEQ ID NO: 11 having a length of at least 5 amino acids andat most 60 amino acids or a BoNT/B peptide of SEQ ID NO: 11 having alength of at least 5 amino acids and at most 60 amino acids.

As used herein, the term “non-naturally occurring BoNT/B variant” meansany BoNT/B produced with the aid of human manipulation, including,without limitation, a BoNT/B produced by genetic engineering usingrandom mutagenesis or rational design and a BoNT/B produced by chemicalsynthesis. Non-limiting examples of non-naturally occurring BoNT/Bvariants include, e.g., conservative BoNT/B variants, non-conservativeBoNT/B variants and chimeric BoNT/B peptides.

As used herein, the term “conservative BoNT/B variant” means a BoNT/Bthat has at least one amino acid substituted by another amino acid or anamino acid analog that has at least one property similar to that of theoriginal amino acid from the reference BoNT/B of SEQ ID NO: 1. Examplesof properties include, without limitation, similar size, topography,charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bondingcapacity, hydrogen-bonding capacity, a physicochemical property, of thelike, or any combination thereof. A conservative BoNT/B variant canfunction in substantially the same manner as the reference BoNT/B of SEQID NO: 1 on which the conservative BoNT/B variant is based, and can besubstituted for the reference BoNT/B of SEQ ID NO: 1 in any aspect ofthe present invention. A conservative BoNT/B variant may substitute oneor more amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, ten or more aminoacids, 20 or more amino acids, 30 or more amino acids, 40 or more aminoacids, 50 or more amino acids, 100 or more amino acids, 200 or moreamino acids, 300 or more amino acids, 400 or more amino acids, or 500 ormore amino acids from the reference BoNT/B of SEQ ID NO: 1 on which theconservative BoNT/B variant is based. A conservative BoNT/B variant canalso substitute at least 10 contiguous amino acids, at least 15contiguous amino acids, at least 20 contiguous amino acids, or at least25 contiguous amino acids from the reference BoNT/B of SEQ ID NO: 1 onwhich the conservative BoNT/B variant is based, that possess at least50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference BoNT/B of SEQ ID NO: 1 on which the conservative BoNT/Bvariant is based.

As a non-limiting example, a conservative BoNT/B variant can be asequence in which a first uncharged polar amino acid is conservativelysubstituted with a second (non-identical) uncharged polar amino acidsuch as cysteine, serine, threonine, tyrosine, glycine, glutamine orasparagine or an analog thereof. A conservative BoNT/B variant also canbe, for example, a sequence in which a first basic amino acid isconservatively substituted with a second basic amino acid such asarginine, lysine, histidine, 5-hydroxylysine, N-methyllysine or ananalog thereof. Similarly, a conservative BoNT/B variant can be, forexample, a sequence in which a first hydrophobic amino acid isconservatively substituted with a second hydrophobic amino acid such asalanine, valine, leucine, isoleucine, proline, methionine, phenylalanineor tryptophan or an analog thereof. In the same way, a conservativeBoNT/B variant can be, for example, a sequence in which a first acidicamino acid is conservatively substituted with a second acidic amino acidsuch as aspartic acid or glutamic acid or an analog thereof; a sequencein which an aromatic amino acid such as phenylalanine is conservativelysubstituted with a second aromatic amino acid or amino acid analog, forexample, tyrosine; or a sequence in which a first relatively small aminoacid such as alanine is substituted with a second relatively small aminoacid or amino acid analog such as glycine or valine or an analogthereof.

As a non limiting example, a BoNT/B peptide comprising amino acidsequence 974-992 from a conservative BoNT/B variant can have, e.g.,leucine 975 substituted with isoleucine, isoleucine 976 substituted withleucine, aspartic acid 977 substituted with glutamic acid, isoleucine978 substituted with leucine, lysine 981 substituted with arginine,lysine 983 substituted with arginine, phenylalanine 786 substituted withtyrosine, phenylalanine 787 substituted with tyrosine, glutamic acid 988substituted with aspartic acid, tyrosine 789 substituted withphenylalanine, isoleucine 991 substituted with leucine, arginine 992substituted with lysine, or any combination thereof.

As another non limiting example, a BoNT/B peptide comprising amino acidsequence 736-754 from a conservative BoNT/B variant can have, e.g.,tyrosine 736 substituted with phenylalanine, arginine 737 substitutedwith lysine, tyrosine 738 substituted with phenylalanine, isoleucine 740substituted with leucine, tyrosine 741 substituted with phenylalanine,glutamic acid 743 substituted with aspartic acid, lysine 744 substitutedwith arginine, glutamic acid 745 substituted with aspartic acid, lysine746 substituted with arginine, isoleucine 749 substituted with leucine,isoleucine 751 substituted with leucine, aspactic acid 752 substitutedwith glutamic acid, phenylalanine 753 substituted with tyrosine, or anycombination thereof.

As still another non limiting example, a BoNT/B peptide comprising aminoacid sequence 1058-1076 from a conservative BoNT/B variant can have,e.g., tyrosine 1058 substituted with phenylalanine, phenylalanine 1059substituted with tyrosine, isoleucine 1061 substituted with leucine,phenylalanine 1062 substituted with tyrosine, glutamic acid 1065substituted with aspartic acid, leucine 1066 substituted withisoleucine, isoleucine 1071 substituted with leucine, glutamic acid 1072substituted with aspartic acid, glutamic acid 1073 substituted withaspartic acid, arginine 1074 substituted with lysine, tyrosine 1075substituted with phenylalanine, lysine 1076 substituted with arginine,or any combination thereof.

As yet another non limiting example, a BoNT/B peptide comprising aminoacid sequence 890-908 from a conservative BoNT/B variant can have, e.g.,glutamic acid 892 substituted with aspartic acid, leucine 893substituted with isoleucine, aspartic acid 895 substituted with glutamicacid, lysine 896 substituted with arginine, phenylalanine 899substituted with tyrosine, lysine 900 substituted with arginine, leucine901 substituted with isoleucine, lysine 908 substituted with arginine,or any combination thereof.

As used herein, the term “non-conservative BoNT/B variant” means aBoNT/B in which 1) at least one amino acid is deleted from the referenceBoNT/B of SEQ ID NO: 1 on which the non-conservative BoNT/B variant isbased; 2) at least one amino acid added to the reference BoNT/B of SEQID NO:1 on which the non-conservative BoNT/B is based; or 3) at leastone amino acid is substituted by another amino acid or an amino acidanalog that does not share any property similar to that of the originalamino acid from the reference BoNT/B of SEQ ID NO: 1. A non-conservativeBoNT/B variant can function in substantially the same manner as thereference BoNT/B of SEQ ID NO: 1 on which the non-conservative BoNT/Bvariant is based, and can be substituted for the reference BoNT/B of SEQID NO: 1 in any aspect of the present invention. A non-conservativeBoNT/B variant can delete one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, and ten or more amino acids from the reference BoNT/B ofSEQ ID NO: 1 on which the non-conservative BoNT/B variant is based. Anon-conservative BoNT/B variant can add one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, and ten or more amino acids to the referenceBoNT/B of SEQ ID NO: 1 on which the non-conservative BoNT/B variant isbased. A non-conservative BoNT/B variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, 50or more amino acids, 100 or more amino acids, 200 or more amino acids,300 or more amino acids, 400 or more amino acids, or 500 or more aminoacids from the reference BoNT/B of SEQ ID NO: 1 on which thenon-conservative BoNT/B variant is based. A non-conservative BoNT/Bvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference BoNT/B of SEQ ID NO:1 on which the non-conservative BoNT/B variant is based, that possess atleast 50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference BoNT/B of SEQ ID NO: 1 on which the non-conservative BoNT/Bvariant is based.

A BoNT/B peptide disclosed in the present specification, such as, e.g.,a BoNT/B peptide derived from a naturally occurring BoNT/B or a BoNT/Bpeptide derived from a non-naturally occurring BoNT/B can be operablylinked with another polypeptide to form a chimeric BoNT/B peptide. Asused herein, the term “chimeric BoNT/B peptide” means a moleculecomprising at least a portion of a BoNT/B peptide disclosed in thepresent specification and at least a portion of at least one otherpolypeptide to form a BoNT/B chimeric peptide. In aspects of thisembodiment, a chimeric BoNT/B protein of the invention can have avariety of lengths including, but not limited to, a length of at most100 residues, at most 200 residues, at most 300 residues, at most 400residues, at most 500 residues, at most 800 residues or at most 1000residues. In other aspects of this embodiment, a chimeric BoNT/B proteinof the invention can have a variety of lengths including, but notlimited to, a length of at least 100 residues, at least 200 residues, atleast 300 residues, at least 400 residues, at least 500 residues, atleast 800 residues or at least 1000 residues. Non-limiting examples of achimeric BoNT/B peptide include, e.g., a BoNT/B peptide operably linkedwith a carrier, such as, e.g., keyhole limpet hemacyanin (KLH),ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA),soybean trypsin inhibitor (STI) or multiple attachment peptide (MAP)technology; a BoNT/B peptide operably linked with a with an immunogenicpolypeptide, such as, e.g., flagellin or cholera enterotoxin; a BoNT/Bpeptide operably linked with a with an immunomodulatory polypeptide,such as, e.g., IL-2 or B7-1; a BoNT/B peptide operably linked with awith tolerogenic polypeptide, such as, e.g., another BoNT/B peptide oran antibody selectively reactive with interleukin-12; and a BoNT/Bpeptide operably linked with a synthetic peptide sequence.

As used herein, the term “amino acid” means both naturally occurring andnon-naturally occurring amino acids as well as amino acid analogs andmimetics, and includes, but is not limited to, alanyl, valinyl,leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, andhistidinyl. As such, a BoNT/B peptide derived from, e.g., a naturallyoccurring BoNT/B or a non-naturally occurring BoNT/B, can contain one ormore non-amide linkage substitutions between amino acids, one or morenaturally occurring amino acid substitutions, one or more non-naturallyoccurring amino acid substitutions, one or more amino acid analogsubstitutions, or one or more mimetic substitutions.

As used herein, the term “naturally occurring amino acid substitution”when used in reference to a BoNT/B means a BoNT/B peptide that has beenaltered from the BoNT/B peptide of SEQ ID NO: 1 in which a first aminoacid from the BoNT/B peptide of SEQ ID NO: 1 is substituted by anaturally occurring amino acid that has at least one property similar tothat of the first amino acid. Naturally occurring amino acids includethe 20 (L)-amino acids utilized during protein biosynthesis as well asothers such as, without limitation, 4-hydroxyproline, hydroxylysine,desmosine, isodesmosine, homocysteine, citrulline and ornithine.

As used herein, the term “non-naturally occurring amino acidsubstitution” when used in reference to a BoNT/B means a BoNT/B peptidethat has been altered from the BoNT/B peptide of SEQ ID NO: 1 in which afirst amino acid from the BoNT/B peptide of SEQ ID NO: 1 is substitutedby a non-naturally occurring amino acid that has at least one propertysimilar to that of the first amino acid. Examples of non-naturallyoccurring amino acids, include, without limitation, (D)-amino acids,norleucine, norvaline, p-fluorophenylalanine, ethionine and the like.

As used herein, “amino acid analog substitution” when used in referenceto a BoNT/B means a BoNT/B peptide that has been altered from the BoNT/Bpeptide of SEQ ID NO: 1 in which a first amino acid from the BoNT/Bpeptide of SEQ ID NO: 1 is substituted by a modified natural ornon-natural amino acid that has at least one property similar to that ofthe first amino acid. Examples of modifications to either a naturallyoccurring amino acid or a non-naturally occurring amino acid, include,without limitation, substitution or replacement of chemical groups ormoieties on the amino acid or by derivitization of the amino acid. ABoNT/B amino acid analog can function in substantially the same manneras the BoNT/B peptide of SEQ ID NO: 1 and can be substituted for theBoNT/B peptide of SEQ ID NO: 1 in any aspect of the present invention. ABoNT/B amino acid analog may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids, 50 or more aminoacids from the BoNT/B peptide of SEQ ID NO: 1, or a portion thereof.

As used herein, the term “mimetic substitution” when used in referenceto a BoNT/B means a BoNT/B peptide that has been altered from the BoNT/Bpeptide of SEQ ID NO: 1 in which a first amino acid from the BoNT/Bpeptide of SEQ ID NO: 1 is substituted by a non-natural structure thathas at least one property similar to that of the first amino acid.Examples of mimetic properties include, without limitation, topographyof a peptide primary structural element, functionality of a peptideprimary structural element, topology of a peptide secondary structuralelement, functionality of a peptide secondary structural element, of thelike, or any combination thereof. A BoNT/B mimetic can function insubstantially the same manner as the BoNT/B peptide of SEQ ID NO: 1 andcan be substituted for the BoNT/B peptide of SEQ ID NO: 1 in any aspectof the present invention. A BoNT/B mimetic may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, 50or more amino acids from the BoNT/B peptide of SEQ ID NO: 1, or aportion thereof. As an example, an organic structure that mimicsarginine can have a positive charge moiety located in similar molecularspace and having the same degree of mobility as the e-amino group of theside chain of the naturally occurring arginine amino acid.

Non-limiting examples of specific protocols for making and usingnaturally occurring amino acids, non-naturally occurring amino acids,amino acid analogs and mimetics are described in, e.g., John Jones,AMINO ACID PEPTIDE SYNTHESIS (Oxford University Press, 2 ed., 2002);Roberts and Vellaccio, p. 341 (THE PEPTIDES: ANALYSIS, SYNTHESIS,BIOLOGY Vol. 5, Erhard Gross & Johannes Meinhofer, eds., Academic Press,Inc., 1983); Mark J. Suto et al., Cytokine Restraining Agents, U.S. Pat.No. 5,420,109 (May 30, 1995); Chapter 7 of Bodanzsky, PRINCIPLES OFPEPTIDE SYNTHESIS (Springer-Verlag, 2^(nd) ed. 1993); Stewart and YoungSOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., 2 ed. 1984); FMOCSOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (Weng C. Chan &Peter D. White eds., Oxford University Press, 2000); Amy S. Ripka &Daniel H. Rich, Peptidomimetic design, 2(4) CURR. OPIN. CHEM. BIOL.441-452 (1998); and M. Angels Estiarte & Daniel H. Rich, Peptidomimeticsfor Drug Design, 803-861 (BURGER'S MEDICINAL CHEMISTRY AND DRUGDISCOVERY Vol. 1 PRINCIPLE AND PRACTICE, Donald J. Abraham ed.,Wiley-Interscience, 6^(th) ed 2003). One skilled in the art understandsthat these and other well known amino acid analogs and mimetics can beuseful in the BoNT/B peptides of the invention.

In is envisioned that any and all procedures that can synthesis a BoNT/Bpeptide disclosed in the present specification can be used. As anon-limiting example, a BoNT/B peptide disclosed in the presentspecification can be produced by chemical synthesis using an automaticpeptide synthesizer employing the chemistry provided by themanufacturer, such as, e.g., a Model 430A automatic peptide synthesizeror a 431A automatic peptide synthesizer (Applied Biosystems, Inc.,Foster City, Calif.). Methods for synthesizing peptides are well knownin the art, see, e.g., Bodanzsky, Principles of Peptide Synthesis (1sted. & 2d rev. ed.), Springer-Verlag, New York, N.Y. (1984 & 1993), andChapter 7; Stewart and Young, Solid Phase Peptide Synthesis, (2d ed.),Pierce Chemical Co., Rockford, Ill. (1984).

Thus, in an embodiment, a BoNT/B peptide has a length of at least 5amino acids and at most 60 amino acids. In an aspect of this embodiment,a BoNT/B peptide of SEQ ID NO: 1 has a length of at least 5 amino acidsand at most 60 amino acids. In other aspects of this embodiment, aBoNT/B peptide comprises an amino acid sequence selected from the groupconsisting of amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO: 5, aminoacids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ ID NO: 3,amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO:7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 of SEQ IDNO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 ofSEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1, amino acids932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO: 1, aminoacids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ ID NO: 7,amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO:3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ IDNO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 ofSEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, aminoacids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7,amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 of SEQ IDNO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 ofSEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, amino acids1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQ ID NO:7.

In other aspects of this embodiment, a BoNT/B peptide comprises an aminoacid sequence selected from the group consisting of amino acids 736-754of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO: 5, aminoacids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3,amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO:7, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ IDNO: 5, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1058-1076 ofSEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, aminoacids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3,amino acids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptide comprises anamino acid sequence selected from the group consisting of amino acids616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ IDNO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 ofSEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

In another embodiment, a BoNT/B peptide has a length of at least 5 aminoacids and at most 60 amino acids and is derived from a naturallyoccurring BoNT/B. In aspects of this embodiment, the naturally occurringBoNT/B is a BoNT/B isoform or a BoNT/B subtype.

In another embodiment, a BoNT/B peptide has a length of at least 5 aminoacids and at most 60 amino acids and is derived from a non-naturallyoccurring BoNT/B. In aspects of this embodiment, the naturally occurringBoNT/B is a conservative BoNT/B variant or a non-conservative BoNT/Bvariant.

In aspects of this embodiment, a BoNT/B peptide comprises, e.g., 1-4conservative amino acid substitutions to amino acids 610-628 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids 736-754of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 736-754 of SEQ ID NO: 5, 1-4 conservative amino acid substitutionsto amino acids 778-796 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 820-838 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 820-838 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 820-838of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 820-838 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 862-880 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 862-880 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 890-908 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 890-908of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 890-908 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 918-936 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 918-936 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 932-950 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 932-950 of SEQ IDNO: 5, 1-4 conservative amino acid substitutions to amino acids 960-978of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 960-978 of SEQ ID NO: 3, 1-4 conservative amino acid substitutionsto amino acids 960-978 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 974-992 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1030-1048 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 1030-1048 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 1030-1048 of SEQ ID NO: 7, 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids1058-1076 of SEQ ID NO: 3, 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 5, 1-4 conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 1072-1090 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1072-1090 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1072-1090 of SEQ ID NO: 7, 1-4 conservative amino acid substitutions toamino acids 1254-1272 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 1254-1272 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 1268-1291 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1268-1291 of SEQ ID NO: 5 or 1-4 conservative amino acid substitutionsto amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/B peptide comprises, e.g.,1-4 conservative amino acid substitutions to amino acids 736-754 of SEQID NO: 1, 1-4 conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 5, 1-4 conservative amino acid substitutions toamino acids 778-796 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 890-908 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 890-908of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 890-908 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 974-992 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 974-992of SEQ ID NO: 7, 1-4 conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 1058-1076 of SEQ ID NO: 5, 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1268-1291 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 5 or 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 7.

In other aspects of this embodiment, a BoNT/B peptide comprises, e.g.,1-4 conservative amino acid substitutions to amino acids 616-626 of SEQID NO: 1, 1-4 conservative amino acid substitutions to amino acids735-745 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 735-745 of SEQ ID NO: 5, 1-4 conservative amino acidsubstitutions to amino acids 778-789 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 867-877 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 867-877 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids 895-905of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 895-905 of SEQ ID NO: 3, 1-4 conservative amino acid substitutionsto amino acids 929-939 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 974-984 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 974-984 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1039-1049 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1039-1049 of SEQ ID NO: 5, 1-4 conservative amino acidsubstitutions to amino acids 1039-1049 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 1065-1075 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1065-1075 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1065-1075 of SEQ ID NO: 5, 1-4 conservative amino acid substitutions toamino acids 1065-1075 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 1269-1281 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 1269-1281 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 1269-1281 of SEQ IDNO: 5 or 1-4 conservative amino acid substitutions to amino acids1269-1281 of SEQ ID NO: 7.

In still aspects of this embodiment, a BoNT/B peptide comprises, e.g.,1-4 non-conservative amino acid substitutions to amino acids 610-628 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 736-754 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 736-754 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 778-796 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids778-796 of SEQ ID NO: 5, 1-4 non-conservative amino acid substitutionsto amino acids 820-838 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 820-838 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 820-838 of SEQID NO: 5, 1-4 non-conservative amino acid substitutions to amino acids820-838 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 862-880 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 862-880 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 3, 1-4 non-conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 918-936 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids918-936 of SEQ ID NO: 3, 1-4 non-conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 960-978 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids960-978 of SEQ ID NO: 3, 1-4 non-conservative amino acid substitutionsto amino acids 960-978 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids974-992 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 1030-1048 of SEQ ID NO: 1, 1-4 non-conservative aminoacid substitutions to amino acids 1030-1048 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1030-1048 ofSEQ ID NO: 5, 1-4 non-conservative amino acid substitutions to aminoacids 1030-1048 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 1072-1090 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 1072-1090 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 1072-1090 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 1254-1272 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 1254-1272 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 5 or 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptide comprises,e.g., 1-4 non-conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 736-754 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 778-796 of SEQID NO: 5, 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids974-992 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 1058-1076 of SEQ ID NO: 1, 1-4 non-conservative aminoacid substitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 5, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 5 or 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptide comprises,e.g., 1-4 non-conservative amino acid substitutions to amino acids616-626 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 735-745 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 735-745 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 778-789 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids867-877 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 867-877 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 895-905 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 895-905 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids929-939 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 974-984 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 974-984 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids1039-1049 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 1039-1049 of SEQ ID NO: 5, 1-4 non-conservative aminoacid substitutions to amino acids 1039-1049 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 1065-1075 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 1065-1075 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 7, 1-4 non-conservative amino acid substitutions to aminoacids 1269-1281 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1269-1281 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1269-1281 ofSEQ ID NO: 5 or 1-4 non-conservative amino acid substitutions to aminoacids 1269-1281 of SEQ ID NO: 7.

A BoNT/B peptide disclosed in the present specification has a length ofat least five amino acids and at most 60 amino acids. Such a BoNT/Bpeptide can comprise an immunoreactive BoNT/B fragment. As used herein,the term “immunoreactive BoNT/B fragment” when used in reference to aBoNT/B means a portion of a BoNT/B peptide capable of selectivelybinding to an anti-BoNT/B antibody. As used herein, the term“selectively” means having a unique effect or influence or reacting inonly one way or with only one thing. An immunoreactive BoNT/B fragmentcan function in substantially the same manner as the BoNT/B peptide ofSEQ ID NO: 1 and can be substituted for the BoNT/B peptide of SEQ ID NO:1 in any aspect of the present invention. An immunoreactive BoNT/Bfragment can function in substantially the same manner as the referenceBoNT/B peptide on which the immunoreactive BoNT/B fragment is based, andcan be substituted for the reference BoNT/B peptide in any aspect of thepresent invention. An immunoreactive BoNT/B fragment is capable ofselective binding to anti-BoNT/B antibodies from one or more species. Animmunoreactive BoNT/B fragment of a BoNT/B peptide generally has from atleast six amino acids to at most 60 amino acids. An immunoreactiveBoNT/B fragment of a BoNT/B peptide can have a length of, e.g., at least6 amino acids, at least 7 amino acids, at least 8 amino acids, at least9 amino acids, at least 10 amino acids, at least 12 amino acids, atleast 15 amino acids, at least 18 amino acids, at least 20 amino acids,at least 25 amino acids at least 30 amino acids or at least 35 aminoacids. An immunoreactive fragment of a BoNT/B peptide also can have alength of, e.g., at most 6 amino acids, at most 7 amino acids, at most 8amino acids, at most 9 amino acids, at most 10 amino acids, at most 12amino acids, at most 15 amino acids, at most 18 amino acids, at most 20amino acids, at most 25 amino acids, at most 30 amino acids or at most35 amino acids. An immunoreactive BoNT/B fragment disclosed in thepresent specification can comprise at least six consecutive amino acidsor can comprise at least six non-consecutive amino acids and can haveany number of conservative, non-conservative, analog or mimetic aminoacid substitutions, and the like, as disclosed in the presentspecification.

An immunoreactive fragment can be identified using any of a variety ofroutine assays for detecting peptide antigen-antibody complexes, thepresence of which is an indicator of selective binding. Such assaysinclude, without limitation, enzyme-linked immunosorbent assays,radioimmunoassays, western blotting, enzyme immunoassays, fluorescenceimmunoassays, luminescent immunoassays and the like and generally areequivalent to the radioimmunoassay disclosed herein in Example 2.Methods for detecting a complex between a peptide and an antibody, andthereby determining if the peptide is an immunoreactive fragment arewell known to those skilled in the art and are described, e.g., inANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane, eds., ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1998a); and USING ANTIBODIES:A LABORATORY MANUAL: PORTABLE PROTOCOL No. I (Edward Harlow & DavidLane, Cold Spring Harbor Laboratory Press, 1998b).

Thus, in an embodiment, an immunoreactive fragment of a BoNT/B peptidehas a length of at least five amino acids and at most 60 amino acids. Inaspects of this embodiment, an immunoreactive fragment of a BoNT/Bpeptide comprises, e.g., at least 6 consecutive amino acids, at least 7consecutive amino acids, at least 8 consecutive amino acids, at least 9consecutive amino acids, at least 10 consecutive amino acids, at least12 consecutive amino acids, at least 15 consecutive amino acids, atleast 18 consecutive amino acids or at least 20 consecutive amino acids.In other aspects of this embodiment, an immunoreactive fragment of aBoNT/B peptide comprises, e.g., at most 6 consecutive amino acids, atmost 7 consecutive amino acids, at most 8 consecutive amino acids, atmost 9 consecutive amino acids, at most 10 consecutive amino acids, atmost 12 consecutive amino acids, at most 15 consecutive amino acids, atmost 18 consecutive amino acids or at most 20 consecutive amino acids.

In still other aspects of this embodiment, an immunoreactive fragment ofa BoNT/B peptide comprises, e.g., at least 6 non-consecutive aminoacids, at least 7 non-consecutive amino acids, at least 8non-consecutive amino acids, at least 9 non-consecutive amino acids, atleast 10 non-consecutive amino acids, at least 12 non-consecutive aminoacids, at least 15 non-consecutive amino acids, at least 18non-consecutive amino acids or at least 20 non-consecutive amino acids.In still other aspects of this embodiment, an immunoreactive fragment ofa BoNT/B peptide comprises, e.g., at most 6 non-consecutive amino acids,at most 7 non-consecutive amino acids, at most 8 non-consecutive aminoacids, at most 9 non-consecutive amino acids, at most 10 non-consecutiveamino acids, at most 12 non-consecutive amino acids, at most 15non-consecutive amino acids, at most 18 non-consecutive amino acids orat most 20 non-consecutive amino acids.

In still other aspects of this embodiment, an immunoreactive fragment ofa BoNT/B peptide can comprise, e.g., from five to sixty amino acids,from five to fifty amino acids, from eight to fifty amino acids, fromten to fifty amino acids, from five to twenty amino acids, from eight totwenty amino acids, from ten to twenty amino acids, from twelve totwenty amino acids or from fifteen to twenty amino acids.

In another embodiment, a BoNT/B peptide comprises an immunogenic BoNT/Bfragment. In an aspect of this embodiment, an immunogenic BoNT/Bfragment comprises at least six consecutive amino acids of a BoNT/Bpeptide. In other aspects of this embodiment, an immunogenic BoNT/Bfragment comprises at least six consecutive amino acids from, e.g.,amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO:1, amino acids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ IDNO: 1, amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQID NO: 1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838 ofSEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880of SEQ ID NO: 1, amino acids 862-880 of SEQ ID NO: 7, amino acids890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3, aminoacids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 7,amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ ID NO:3, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ IDNO: 5, amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQID NO: 3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992 ofSEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, aminoacids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1,amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ IDNO: 5, amino acids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 ofSEQ ID NO: 1, amino acids 1072-1090 of SEQ ID NO: 3, amino acids1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1, aminoacids 1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1,amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six consecutive amino acids from, e.g., amino acids616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ IDNO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 ofSEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

In still another aspect of this embodiment, an immunogenic BoNT/Bfragment comprises at least six non-consecutive amino acids of a BoNT/Bpeptide. In still other aspects of this embodiment, an immunogenicBoNT/B fragment comprises at least six non-consecutive amino acids from,e.g., amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ IDNO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQID NO: 1, amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 ofSEQ ID NO: 1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids862-880 of SEQ ID NO: 1, amino acids 862-880 of SEQ ID NO: 7, aminoacids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3,amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO:7, amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ IDNO: 3, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQID NO: 5, amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 ofSEQ ID NO: 3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, aminoacids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5,amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ IDNO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 ofSEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, amino acids1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 of SEQ ID NO: 3, aminoacids 1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1,amino acids 1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 1, amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 ofSEQ ID NO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, an immunogenic BoNT/Bfragment comprises at least six non-consecutive amino acids from, e.g.,amino acids 616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO:1, amino acids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ IDNO: 1, amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQID NO: 7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 ofSEQ ID NO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984of SEQ ID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids974-984 of SEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, aminoacids 1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7,amino acids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ IDNO: 3, amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 ofSEQ ID NO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 oramino acids 1269-1281 of SEQ ID NO: 7.

The present invention provides, in part, a BoNT/B peptide composition.It is envisioned that a BoNT/B peptide composition can comprise any ofthe BoNT/B peptides disclosed in the present specification, including,without limitation, a BoNT/B peptide derived from a naturally occurringBoNT/B, such as, e.g., the BoNT/B of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, aBoNT/B isoform or a BoNT/B subtype; and a BoNT/B peptide derived from anon-naturally occurring BoNT/B, such as, e.g., a conservative BoNT/Bvariant, a non-conservative BoNT/B variant and a chimeric BoNT/Bpeptide. BoNT/B peptides disclosed in the present specification can beselected, for example, depending on immunological factors, such aspotency of the peptide in inducing an immune response, and technicalfactors, such as chemical synthesis yields. It is also understood thatthe two or more different BoNT/B peptides can be provided separately oras part of a compound molecule such as a chimeric peptide orheterologous protein. Examples of BoNT/B peptide compositions, include,without limitation, tolerogizing compositions comprising a tolerogizingagent and a BoNT/B peptide and immune response inducing compositionscomprising an adjuvant and a BoNT/B peptide.

In an embodiment, a BoNT/B peptide composition can comprise one BoNT/Bpeptide disclosed in the present specification. In another embodiment, aBoNT/B peptide composition can comprise a plurality of different BoNT/Bpeptides disclosed in the present specification. Thus, in aspects ofthis embodiment, a BoNT/B peptide composition comprises one or moredifferent BoNT/B peptides, two or more different BoNT/B peptides, threeor more different BoNT/B peptides, four or more different BoNT/Bpeptides, five or more different BoNT/B peptides, six or more differentBoNT/B peptides, seven or more different BoNT/B peptides, eight or moredifferent BoNT/B peptides, nine or more different BoNT/B peptides, tenor more different BoNT/B peptides, 15 or more different BoNT/B peptidesor 20 or more different BoNT/B peptides.

In other aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more different BoNT/B peptides derived from the BoNT/Bof SEQ ID NO: 1, the BoNT/B of SEQ ID NO: 3, the BoNT/B of SEQ ID NO: 5,the BoNT/B of SEQ ID NO: 7, the BoNT/B of SEQ ID NO: 9, the BoNT/B ofSEQ ID NO: 11 or the BoNT/B of SEQ ID NO: 13.

In still other aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more different BoNT/B peptides comprising an amino acidsequence selected from the group consisting of amino acids 610-628 ofSEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 1, aminoacids 820-838 of SEQ ID NO: 3, amino acids 820-838 of SEQ ID NO: 5,amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880 of SEQ ID NO:1, amino acids 862-880 of SEQ ID NO: 7, amino acids 890-908 of SEQ IDNO: 1, amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQID NO: 5, amino acids 890-908 of SEQ ID NO: 7, amino acids 918-936 ofSEQ ID NO: 1, amino acids 918-936 of SEQ ID NO: 3, amino acids 932-950of SEQ ID NO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQ ID NO: 3, aminoacids 960-978 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1,amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO:7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, aminoacids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 of SEQ ID NO: 1,amino acids 1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 of SEQ IDNO: 7, amino acids 1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 ofSEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1, amino acids1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5 andamino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more different BoNT/B peptides, each comprising anamino acid sequence selected from the group consisting of amino acids616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ IDNO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 ofSEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

In another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-992 ofSEQ ID NO: 1. In another aspect of this embodiment, a BoNT/B peptidecomposition comprises a BoNT/B peptide comprising amino acid sequence is736-754 of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/Bpeptide composition comprises a BoNT/B peptide comprising amino acidsequence is 1058-1076 of SEQ ID NO: 1. In another aspect of thisembodiment, a BoNT/B peptide composition comprises a BoNT/B peptidecomprising amino acid sequence is 890-908 of SEQ ID NO: 1. In anotheraspect of this embodiment, a BoNT/B peptide composition comprises aBoNT/B peptide comprising amino acid sequence is 778-796 of SEQ IDNO: 1. In another aspect of this embodiment, a BoNT/B peptidecomposition comprises a BoNT/B peptide comprising amino acid sequence is932-950 of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/Bpeptide composition comprises a BoNT/B peptide comprising amino acidsequence is 1268-1291 of SEQ ID NO: 1.

In another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 616-626 ofSEQ ID NO: 1. In another aspect of this embodiment, a BoNT/B peptidecomposition comprises a BoNT/B peptide comprising amino acid sequence is735-745 of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/Bpeptide composition comprises a BoNT/B peptide comprising amino acidsequence is 778-789 of SEQ ID NO: 1.

In another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 867-877 ofSEQ ID NO: 1. In another aspect of this embodiment, a BoNT/B peptidecomposition comprises a BoNT/B peptide comprising amino acid sequence is895-905 of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/Bpeptide composition comprises a BoNT/B peptide comprising amino acidsequence is 929-939 of SEQ ID NO: 1. In another aspect of thisembodiment, a BoNT/B peptide composition comprises a BoNT/B peptidecomprising amino acid sequence is 974-984 of SEQ ID NO: 1. In anotheraspect of this embodiment, a BoNT/B peptide composition comprises aBoNT/B peptide comprising amino acid sequence is 1039-1049 of SEQ IDNO: 1. In another aspect of this embodiment, a BoNT/B peptidecomposition comprises a BoNT/B peptide comprising amino acid sequence is1065-1075 of SEQ ID NO: 1. In another aspect of this embodiment, aBoNT/B peptide composition comprises a BoNT/B peptide comprising aminoacid sequence is 1269-1281 of SEQ ID NO: 1.

In yet another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-992 ofSEQ ID NO:1 and a BoNT/B peptide comprising amino acid sequence is736-754 of SEQ ID NO: 1. In yet another aspect of this embodiment, aBoNT/B peptide composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-992 of SEQ ID NO:1 and a BoNT/B peptide comprisingamino acid sequence is 1058-1076 of SEQ ID NO: 1. In yet another aspectof this embodiment, a BoNT/B peptide composition comprises a BoNT/Bpeptide comprising amino acid sequence is 974-992 of SEQ ID NO:1 and aBoNT/B peptide comprising amino acid sequence is 890-908 of SEQ ID NO:1.

In yet another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-984 ofSEQ ID NO:1 and a BoNT/B peptide comprising amino acid sequence is735-745 of SEQ ID NO: 1. In yet another aspect of this embodiment, aBoNT/B peptide composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-984 of SEQ ID NO:1 and a BoNT/B peptide comprisingamino acid sequence is 1065-1075 of SEQ ID NO: 1. In yet another aspectof this embodiment, a BoNT/B peptide composition comprises a BoNT/Bpeptide comprising amino acid sequence is 974-984 of SEQ ID NO:1 and aBoNT/B peptide comprising amino acid sequence is 895-905 of SEQ ID NO:1.

In still another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-992 ofSEQ ID NO:1, a BoNT/B peptide comprising amino acid sequence is 736-754of SEQ ID NO: 1 and a BoNT/B peptide comprising amino acid sequence is1058-1076 of SEQ ID NO: 1. In still another aspect of this embodiment, aBoNT/B peptide composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-992 of SEQ ID NO:1, a BoNT/B peptide comprisingamino acid sequence is 736-754 of SEQ ID NO: 1 and a BoNT/B peptidecomprising amino acid sequence is 890-908 of SEQ ID NO: 1.

In still another aspect of this embodiment, a BoNT/B peptide compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-984 ofSEQ ID NO:1, a BoNT/B peptide comprising amino acid sequence is 735-745of SEQ ID NO: 1 and a BoNT/B peptide comprising amino acid sequence is1065-1075 of SEQ ID NO: 1. In still another aspect of this embodiment, aBoNT/B peptide composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-984 of SEQ ID NO:1, a BoNT/B peptide comprisingamino acid sequence is 735-745 of SEQ ID NO: 1 and a BoNT/B peptidecomprising amino acid sequence is 895-905 of SEQ ID NO: 1.

In another embodiment, a BoNT/B peptide composition comprises one ormore different BoNT/B peptides derived from a non-naturally occurringBoNT/B. In aspects of this embodiment, the non-naturally occurringBoNT/B is a conservative BoNT/B variant or a non-conservative BoNT/Bvariant.

In aspects of this embodiment, a BoNT/B peptide composition comprisesone or more different BoNT/B peptides derived from a conservative BoNT/Bvariant, two or more different BoNT/B peptides derived from aconservative BoNT/B variant, three or more different BoNT/B peptidesderived from a conservative BoNT/B variant, four or more differentBoNT/B peptides derived from a conservative BoNT/B variant, five or moredifferent BoNT/B peptides derived from a conservative BoNT/B variant,six or more different BoNT/B peptides derived from a conservative BoNT/Bvariant, seven or more different BoNT/B peptides derived from aconservative BoNT/B variant, eight or more different BoNT/B peptidesderived from a conservative BoNT/B variant, nine or more differentBoNT/B peptides derived from a conservative BoNT/B variant, ten or moredifferent BoNT/B peptides derived from a conservative BoNT/B variant, 15or more different BoNT/B peptides derived from a conservative BoNT/Bvariant or 20 or more different BoNT/B peptides derived from aconservative BoNT/B variant.

In aspects of this embodiment, a BoNT/B peptide composition comprisesone or more BoNT/B peptides selected from the group consisting of aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 610-628 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 736-754 of SEQ IDNO: 1, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids820-838 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4 conservativeamino acid substitutions to amino acids 862-880 of SEQ ID NO: 1, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 890-908 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 918-936 of SEQ IDNO: 1, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids960-978 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4 conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 1, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 1030-1048 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 1, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 1072-1090 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids1254-1272 of SEQ ID NO: 1, and a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 1.

In aspects of this embodiment, a BoNT/B peptide composition comprisesone or more BoNT/B peptides selected from the group consisting of aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 736-754 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 778-796 of SEQ IDNO: 1, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4 conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 1, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 1, and a BoNT/B peptide comprising1-4 conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 1.

In further aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more BoNT/B peptides selected from the group consistingof a BoNT/B peptide comprising 1-4 conservative amino acid substitutionsto amino acids 616-626 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 735-745 of SEQ IDNO: 1, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 778-789 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids867-877 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4 conservativeamino acid substitutions to amino acids 895-905 of SEQ ID NO: 1, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 929-939 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 974-984 of SEQ IDNO: 1, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 1039-1049 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids1065-1075 of SEQ ID NO: 1 or a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 1269-1281 of SEQ IDNO: 1.

In further aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more different BoNT/B peptides derived from anon-conservative BoNT/B variant, two or more different BoNT/B peptidesderived from a non-conservative BoNT/B variant, three or more differentBoNT/B peptides derived from a non-conservative BoNT/B variant, four ormore different BoNT/B peptides derived from a non-conservative BoNT/Bvariant, five or more different BoNT/B peptides derived from anon-conservative BoNT/B variant, six or more different BoNT/B peptidesderived from a non-conservative BoNT/B variant, seven or more differentBoNT/B peptides derived from a non-conservative BoNT/B variant, eight ormore different BoNT/B peptides derived from a non-conservative BoNT/Bvariant, nine or more different BoNT/B peptides derived from anon-conservative BoNT/B variant, ten or more different BoNT/B peptidesderived from a non-conservative BoNT/B variant, 15 or more differentBoNT/B peptides derived from a non-conservative BoNT/B variant or 20 ormore different BoNT/B peptides derived from a non-conservative BoNT/Bvariant.

In further aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more BoNT/B peptides selected from the group consistingof a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 610-628 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 778-796 of SEQID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 820-838 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids862-880 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 918-936 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 960-978 of SEQID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative aminoacid substitutions to amino acids 1072-1090 of SEQ ID NO: 1, a BoNT/Bpeptide comprising 1-4 non-conservative amino acid substitutions toamino acids 1254-1272 of SEQ ID NO: 1, and a BoNT/B peptide comprising1-4 non-conservative amino acid substitutions to amino acids 1268-1291of SEQ ID NO: 1.

In further aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more BoNT/B peptides selected from the group consistingof a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 736-754 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids778-796 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids974-992 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 1, and a BoNT/B peptide comprising 1-4 non-conservative aminoacid substitutions to amino acids 1268-1291 of SEQ ID NO: 1.

In further other aspects of this embodiment, a BoNT/B peptidecomposition comprises one or more BoNT/B peptides selected from thegroup consisting of a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 616-626 of SEQ ID NO: 1, aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 735-745 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 778-789 of SEQID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 867-877 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids895-905 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 929-939 of SEQID NO: 1, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 1, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids1039-1049 of SEQ ID NO: 1, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 1 or a BoNT/B peptide comprising 1-4 non-conservative aminoacid substitutions to amino acids 1269-1281 of SEQ ID NO: 1.

In still other aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more different BoNT/B peptides each peptide comprisinga different BoNT/B immunoreactive fragment, two or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment, three or more different BoNT/B peptides each peptidecomprising a different BoNT/B immunoreactive fragment, four or moredifferent BoNT/B peptides each peptide comprising a different BoNT/Bimmunoreactive fragment, five or more different BoNT/B peptides eachpeptide comprising a different BoNT/B immunoreactive fragment, six ormore different BoNT/B peptides each peptide comprising a differentBoNT/B immunoreactive fragment, seven or more different BoNT/B peptideseach peptide comprising a different BoNT/B immunoreactive fragment,eight or more different BoNT/B peptides each peptide comprising adifferent BoNT/B immunoreactive fragment, nine or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment, ten or more different BoNT/B peptides each peptide comprisinga different BoNT/B immunoreactive fragment, 15 or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment, 20 or more different BoNT/B peptides each peptide comprising adifferent BoNT/B immunoreactive fragment, 25 or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment or 30 or more different BoNT/B peptides each peptide comprisinga different BoNT/B immunoreactive fragment.

In other aspects of this embodiment, a BoNT/B peptide compositioncomprises one or more BoNT/B peptides, each peptide comprising adifferent BoNT/B immunoreactive fragment selected from the groupconsisting of at least six consecutive amino acids of 610-628 of SEQ IDNO: 1, at least six consecutive amino acids of 736-754 of SEQ ID NO: 1,at least six consecutive amino acids of 778-796 of SEQ ID NO: 1, atleast six consecutive amino acids of 820-838 of SEQ ID NO: 1, at leastsix consecutive amino acids of 862-880 of SEQ ID NO: 1, at least sixconsecutive amino acids of 890-908 of SEQ ID NO: 1, at least sixconsecutive amino acids of 918-936 of SEQ ID NO: 1, at least sixconsecutive amino acids of 932-950 of SEQ ID NO: 1, at least sixconsecutive amino acids of 960-978 of SEQ ID NO: 1, at least sixconsecutive amino acids of 974-992 of SEQ ID NO: 1, at least sixconsecutive amino acids of 1030-1048 of SEQ ID NO: 1, at least sixconsecutive amino acids of 1058-1076 of SEQ ID NO: 1, at least sixconsecutive amino acids of 1072-1090 of SEQ ID NO: 1, at least sixconsecutive amino acids of 1254-1272 of SEQ ID NO: 1, or at least sixconsecutive amino acids of 1268-1291 of SEQ ID NO: 1. In other aspectsof this embodiment, a BoNT/B peptide composition comprises two or moreBoNT/B peptides, each peptide comprising a different BoNT/Bimmunoreactive fragment selected from the group consisting of at leastsix consecutive amino acids of 616-626 of SEQ ID NO: 1, at least sixconsecutive amino acids of 735-745 of SEQ ID NO: 1, at least sixconsecutive amino acids of 778-789 of SEQ ID NO: 1, at least sixconsecutive amino acids of 867-877 of SEQ ID NO: 1, at least sixconsecutive amino acids of 895-905 of SEQ ID NO: 1, at least sixconsecutive amino acids of 929-939 of SEQ ID NO: 1, at least sixconsecutive amino acids of 974-984 of SEQ ID NO: 1, at least sixconsecutive amino acids of 1039-1049 of SEQ ID NO: 1, at least sixconsecutive amino acids of 1065-1075 of SEQ ID NO: 1 or at least sixconsecutive amino acids of 1269-1281 of SEQ ID NO: 1.

In another embodiment, a BoNT/B peptide composition comprises one ormore BoNT/B peptides, each peptide comprising a different BoNT/Bimmunoreactive fragment selected from the group consisting of at leastsix non-consecutive amino acids of 610-628 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 736-754 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 778-796 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 820-838 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 862-880 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 890-908 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 918-936 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 932-950 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 960-978 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 974-992 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 1030-1048 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 1058-1076 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 1072-1090 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 1254-1272 of SEQ ID NO: 1, or at leastsix non-consecutive amino acids of 1268-1291 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/B peptide composition comprises twoor more BoNT/B peptides, each peptide comprising a different BoNT/Bimmunoreactive fragment selected from the group consisting of at leastsix non-consecutive amino acids of 616-626 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 735-745 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 778-789 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 867-877 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 895-905 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 929-939 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 974-984 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 1039-1049 of SEQ ID NO: 1, at least sixnon-consecutive amino acids of 1065-1075 of SEQ ID NO: 1 or at least sixnon-consecutive amino acids of 1269-1281 of SEQ ID NO: 1.

It is also envisioned that any and all combinations of BoNT/B peptidesdisclosed in the specification can be useful in a BoNT/B peptidecomposition, including, without limitation, a BoNT/B peptide derivedfrom a naturally occurring BoNT/B, such as, e.g., the BoNT/B of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11 or SEQ ID NO: 13, a BoNT/B isoform or a BoNT/B subtype; and aBoNT/B peptide derived from a non-naturally occurring BoNT/B, such as,e.g., a conservative BoNT/B variant, a non-conservative BoNT/B variantand a chimeric BoNT/B peptide. Thus, aspects of this embodiment, aBoNT/B peptide composition comprises, e.g., one or more BoNT/B peptidesderived from a naturally occurring BoNT/B and one or more BoNT/Bpeptides derived from a non-naturally occurring BoNT/B. In other aspectsof this embodiment, a BoNT/B peptide composition comprises, e.g., one ormore BoNT/B peptides comprising one or more BoNT/B peptides derived fromSEQ ID NO: 1 and one or more BoNT/B peptides derived from a conservativeBoNT/B variant; one or more BoNT/B peptides of SEQ ID NO: 1 and one ormore BoNT/B peptides derived from a non-conservative variant; one ormore BoNT/B peptides derived from a conservative BoNT/B variant and oneor more BoNT/B peptides derived from a non-conservative BoNT/B variant;and one or more BoNT/B peptides derived from SEQ ID NO: 1, one or moreBoNT/B peptides derived from a conservative BoNT/B variant and one ormore BoNT/B peptides derived from a non-conservative BoNT/B variant.

Tolerance is an active antigen-dependent process that occurs in anindividual in response to the antigen that results from a previousexposure to the same antigen. Generally speaking, the production ofantibodies by an immune response occurs by a two-step process.Initially, B lymphocytes migrating through the lymphoid tissue areexposed to an antigen whereby these cells become partially activated.Subsequently, if a partially activated B cell encounters a T cell thathas also been activated by the same antigen, antibodies against thatantigen are produced. If the B cell does not receive the appropriatesignal from the corresponding T cell, it will become inactive or die.Immune tolerance is a natural mechanism that eliminates development of Bcells that target “self,” rather than foreign antigens. Therapeuticmethods using tolerogizing compositions can exploit this immunetolerance system. For example, binding of a tolerogizing composition toa specific B cell is thought to stop production of pathogenic antibodiesby causing the inactivatation or death of these pathogenic B cells. Ingeneral, a tolerogizing composition that can be used to tolerize B cellsin an antigen-specific manner lacks the ability to activate T cells, butretains the ability to bind immune B cells. Therefore, an individualsuffering from an immune response to a particular antigen can be treatedwith a tolerogizing composition and become “tolerized” to thatparticular antigen.

The present invention provides, in part, a tolerogizing compositioncomprising a BoNT/B peptide operably linked to a tolerogizing agent.Such tolerogizing compositions are useful for inducing specificimmunological non-reactivity (tolerance) to a botulinum toxin antigen.It is envisioned that any of the BoNT/B peptides disclosed in thepresent specification can be useful in a tolerogizing composition, withthe proviso that the BoNT/B peptide induces a specific immunologicalnon-reactivity (tolerance) to a botulinum toxin antigen. Non-limitingexamples include a BoNT/B peptide derived from a naturally occurringBoNT/B, such as, e.g., the BoNT/B of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, aBoNT/B isoform or a BoNT/B subtype; and a BoNT/B peptide derived from anon-naturally occurring BoNT/B, such as, e.g., a conservative BoNT/Bvariant, a non-conservative BoNT/B variant and a chimeric BoNT/Bpeptide. BoNT/B peptides disclosed in the present specification can beselected, e.g., depending on immunological factors, such as potency ofthe peptide in inducing a tolerogizing response, and technical factors,such as chemical synthesis yields. It is also understood that the two ormore different BoNT/B peptides can be provided separately or as part ofa compound molecule such as a chimeric BoNT/B peptide.

It is envisioned that a wide variety of tolerogizing agents can beuseful in a tolerogizing composition disclosed in the presentspecification. As used herein, the term “tolerogizing agent” means amolecule, compound or polymer that causes, promotes or enhancestolerogenic activity when combined with a BoNT/B peptide disclosed inthe present specification. As non-limiting examples, a tolerogizingagent can be a liquid, solid, or emulsion, depending, for example, onthe route of administration and physical properties of the tolerogizingagent. A tolerogizing agent is operably linked to a BoNT/B peptidedisclosed in the present specification. As used herein, the term“operably linked” when used in reference to a tolerogizing compositionmeans to covalently attach a tolerogizing agent to a BoNT/B peptide in amanner that renders the peptide tolerogenic. Such tolerogizing agentscan be operably linked to a BoNT/B peptide, for example, as described inM. Zouhair Atassi & Tetsuo Ashizawa, PVA or PEG Conjugates of Peptidesfor Epitope-Specific Immunosuppression, U.S. Pat. No. 6,048,529 (Apr.11, 2000); Emilio Barbera-Guillem & M. Bud Nelson, Compositions andMethods for Tolerization in Immune Complex-Mediated Disease Progression,U.S. Pat. No. 6,245,752 (Jun. 12, 2001); and Edward Jess Victoria etal., APL Immunoreactive Peptides, Conjugates Thereof and Methods ofTreatment for APL Antibody-Mediated Pathologies, U.S. Pat. No. 6,410,775(Jun. 25, 2002). A variety of tolerogizing agents are useful in theinvention including, without limitation, polyethylene glycol (PEG),monomethoxypolyethylene glycol (mPEG), and polyvinyl alcohol (PVA).Additional molecules are also known in the art to cause, promote orenhance tolerance, see, e.g., Paul A. Barstad, & Gilbert M. Iverson,Composition For Inducing Humoral Anergy to an Immunogen Comprising a TCell Epitope-Deficient Analog of the Immunogen Conjugated to aNonimmunogenic Carrier, U.S. Pat. No. 5,268,454 (Dec. 7, 1993); M.Zouhair Atassi & Tetsuo Ashizawa, PVA or PEG Conjugates of Peptides forEpitope-Specific Immunosuppression, U.S. Pat. No. 6,048,529 (Apr. 11,2000); and Stephen M. Coutts et al., Composition for Inducing HumoralAnergy to an Immunogen Comprising a T Cell Epitope-Deficient Analog ofthe Immunogen Conjugated to a Nonimmunogenic Valency Platform Molecule,U.S. Pat. No. 6,060,056 (May 9, 2000).

As used herein, the term “tolerogizing response” when used in referenceto a tolerogizing composition comprising a BoNT/B peptide means a BoNT/Bdisclosed in the present specification that has tolerogenic activity asdefined by the ability either alone, or in combination with one or moreother molecules, to produce a decreased immunological response to ananti-BoNT antibody. A BoNT/B peptide exhibiting a tolerogizing responsecan be identified using any of a variety of assays, including in vitroassays such as T-cell proliferation or cytokine secretion assays and invivo assays such as the induction of tolerance in animal models ofbotulinum toxicity. T-cell proliferation assays, for example, are wellrecognized in the art as predictive of tolerogenic activity, see, e.g.,H. Miyahara et al., Identification and Characterization Of A MajorTolerogenic T-Cell Epitope of Type II Collagen That Suppresses Arthritisin B10.RIII Mice, 86(1) IMMUNOLOGY 110-115 (1995); and Knut E. A. Lundinet al, Gliadin-Specific, HLA-DQ(Alpha 1*0501,Beta 1*0201) Restricted TCells Isolated From the Small Intestinal Mucosa of Celiac DiseasePatients, 178(1) J. EXP. MED. 187-196 (1993). A T-cell proliferationassay can be performed, for example, by culturing T-cells withirradiated antigen-presenting cells, such as normal spleen cells, inmicrotiter wells for 3 days with varying concentrations of the BoNT/Bpeptide to be assayed; adding ³H-thymidine; and measuring incorporationof ³H-thymidine into DNA.

A BoNT/B peptide exhibiting a tolerogizing response can also beidentified using a T-cell cytokine secretion assay known in the art. Insuch an assay, T cells can be cultured, for example, with irradiatedantigen-presenting cells in microtiter wells with varying concentrationsof the fragment of interest and, after three days, the culturesupernatants can be assayed for IL-2, IL-4 or IFN-γ as described in C.Czerkinsky et al., Detection of Human Cytokine-Secreting Cells inDistinct Anatomical Compartments, 119 IMMUNOL. REV. 5-22 (1991).

A BoNT/B peptide exhibiting a tolerogizing response can additionally beidentified by its ability to induce tolerance in vivo, as indicated by adecreased immunological response, which can be a decreased T-cellresponse, such as a decreased proliferative response or cytokinesecretion response as described above, or a decreased antibody titer tothe antigen. A neonatal or adult mouse can be tolerized with a fragmentof a BoNT/B peptide, and a T-cell response or anti-BoNT/B antibody titercan be assayed after challenging by immunization. As an example, aneonatal mouse can be tolerized within 48 hours of birth byintraperitoneal administration of about 100 μg of a fragment of a BoNT/Bpeptide emulsified with incomplete Freund's adjuvant and subsequentlyimmunized with BoNT/B at about 8 weeks of age, see, for example,Miyahara et al., supra, 1995. An adult mouse can be tolerizedintravenously with about 0.33 mg of a fragment of a BoNT/B peptide,administered daily for three days (total dose 1 mg), and immunized oneweek later with BoNT/B. A decreased T-cell response, such as decreasedproliferation or cytokine secretion, which indicates tolerogenicactivity, can be measured using T-cells harvested 10 days afterimmunization. In addition, a decreased anti-BoNT/B antibody titer, whichalso indicates tolerogenic activity, can be assayed using bloodharvested 4-8 weeks after immunization. Methods for assaying a T-cellresponse or anti-BoNT/B antibody titer are described above and are wellknown in the art.

Several well-accepted models of botulinum toxicity can be useful inidentifying a BoNT/B peptide exhibiting a tolerogizing response. Suchmodels include, without limitation, rodent, rabbit and monkey models offoodborne botulism, rodent and chicken models of infant botulism androdent models of wound botulism, which are described, for example, inSimpson (Ed.) Botulinum Neurotoxin and Tetanus Toxin Academic Press,Inc., San Diego, Calif. (1989). The skilled person understands thatthese and a variety of other well known in vitro and in vivo assays canbe useful for identifying a tolerogenic fragment of a BoNT/B peptide.

In is envisioned that a tolerogizing composition can also optionallycomprises one or more adjuvants. As used herein, the term “adjuvant”when used in reference to a tolerogizing composition means any substanceor mixture of substances that promotes or enhances tolerogenic activity.A tolerogizing adjuvant can, for example, serve to increase thesolubility of a tolerogizing composition. The use of tolerogizingadjuvants in a tolerogizing composition is well known. Thesetolerogizing adjuvants are diverse in nature. They may, e.g., consist ofliposomes, oily phases, including, without limitation, the Freund typeof adjuvants, such as, e.g., Freund's complete adjuvant (FCA); Freund'sincomplete adjuvant (FIA); sapogenin glycosides, such as, e.g.,saponins; carbopol; N-acetylmuramyl-L-alanyl-D-isoglutamine (commonlyknown as muramyl dipeptide or “MDP”); and lipopolysaccharide (LPS). Suchadjuvants are generally used in the form of an emulsion with an aqueousphase, or, more commonly, may consist of water-insoluble inorganicsalts. These inorganic salts may consist, for example, of aluminumhydroxide, zinc sulfate, colloidal iron hydroxide, calcium phosphate orcalcium chloride. Aluminum hydroxide (Al(OH)₃) is a commonly usedadjuvant. Currently, the only FDA-approved adjuvant for use in humans isaluminum salts (Alum) which are used to “depot” antigens byprecipitation of the antigens. Adjuvants provided above are merelyexemplary. In fact, any tolerogizing adjuvant may be used in atolerogizing composition disclosed in the present specification as longas the adjuvant satisfies the requisite characteristics that arenecessary for practicing the present invention.

Thus, in an embodiment, a tolerogizing composition comprises a BoNT/Bpeptide disclosed in the present specification operably linked to atolerogizing agent. In another embodiment, a tolerogizing compositioncan comprise a plurality of different BoNT/B peptides disclosed in thepresent specification each BoNT/B peptide operably linked to atolerogizing agent. Thus, in aspects of this embodiment, a tolerogizingcomposition comprises one or more different BoNT/B peptides each BoNT/Bpeptide operably linked to a tolerogizing agent, two or more differentBoNT/B peptides each BoNT/B peptide operably linked to a tolerogizingagent, three or more different BoNT/B peptides each BoNT/B peptideoperably linked to a tolerogizing agent, four or more different BoNT/Bpeptides each BoNT/B peptide operably linked to a tolerogizing agent,five or more different BoNT/B peptides each BoNT/B peptide operablylinked to a tolerogizing agent, six or more different BoNT/B peptideseach BoNT/B peptide operably linked to a tolerogizing agent, seven ormore different BoNT/B peptides each BoNT/B peptide operably linked to atolerogizing agent, eight or more different BoNT/B peptides each BoNT/Bpeptide operably linked to a tolerogizing agent, nine or more differentBoNT/B peptides each BoNT/B peptide operably linked to a tolerogizingagent, ten or more different BoNT/B peptides each BoNT/B peptideoperably linked to a tolerogizing agent, 15 or more different BoNT/Bpeptides each BoNT/B peptide operably linked to a tolerogizing agent or20 or more different BoNT/B peptides each BoNT/B peptide operably linkedto a tolerogizing agent.

In aspects of this embodiment, a tolerogizing composition comprises oneor more different BoNT/B peptides comprising an amino acid sequenceselected from the group consisting of amino acids 610-628 of SEQ ID NO:1, amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ IDNO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQID NO: 5, amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838 ofSEQ ID NO: 3, amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838of SEQ ID NO: 7, amino acids 862-880 of SEQ ID NO: 1, amino acids862-880 of SEQ ID NO: 7, amino acids 890-908 of SEQ ID NO: 1, aminoacids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5,amino acids 890-908 of SEQ ID NO: 7, amino acids 918-936 of SEQ ID NO:1, amino acids 918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQ IDNO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQID NO: 1, amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978 ofSEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, aminoacids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7,amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ IDNO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 ofSEQ ID NO: 7, amino acids 1072-1090 of SEQ ID NO: 1, amino acids1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 of SEQ ID NO: 7, aminoacids 1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3,amino acids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ IDNO: 3, amino acids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291of SEQ ID NO: 7.

In other aspects of this embodiment, a tolerogizing compositioncomprises two or more amino acid sequences selected from the groupconsisting of amino acids 616-626 of SEQ ID NO: 1, amino acids 735-745of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5, amino acids778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 1, aminoacids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ ID NO: 1,amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQ ID NO:1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 of SEQ IDNO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049 of SEQID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids 1039-1049of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, amino acids1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5, aminoacids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ ID NO: 1,amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 of SEQ IDNO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

In another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide operably linked to a tolerogizing agent, theBoNT/B peptide comprising an amino acid sequence selected from the groupconsisting of amino acids amino acids 610-628 of SEQ ID NO: 1, aminoacids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5,amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO:5, amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ IDNO: 3, amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQID NO: 7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 ofSEQ ID NO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids890-908 of SEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, aminoacids 918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1,amino acids 932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO:1, amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ IDNO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 ofSEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, aminoacids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3,amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ IDNO: 7, amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 ofSEQ ID NO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3,amino acids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQID NO: 7.

In another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide operably linked to a tolerogizing agent, theBoNT/B peptide comprising an amino acid sequence selected from the groupconsisting of amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids778-796 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 1, aminoacids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5,amino acids 890-908 of SEQ ID NO: 7, amino acids 932-950 of SEQ ID NO:1, amino acids 932-950 of SEQ ID NO: 5, amino acids 974-992 of SEQ IDNO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids1058-1076 of SEQ ID NO: 7, amino acids 1268-1291 of SEQ ID NO: 1, aminoacids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5and amino acids 1268-1291 of SEQ ID NO: 7.

In another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide operably linked to a tolerogizing agent, theBoNT/B peptide comprising an amino acid sequence selected from the groupconsisting of amino acids 616-626 of SEQ ID NO: 1, amino acids 735-745of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5, amino acids778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 1, aminoacids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ ID NO: 1,amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQ ID NO:1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 of SEQ IDNO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049 of SEQID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids 1039-1049of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, amino acids1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5, aminoacids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ ID NO: 1,amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 of SEQ IDNO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

In yet another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-992 ofSEQ ID NO: 1 operably linked to a tolerogizing agent and a BoNT/Bpeptide comprising amino acid sequence is 736-754 of SEQ ID NO: 1operably linked to a tolerogizing agent. In yet another aspect of thisembodiment, a tolerogizing composition comprises a BoNT/B peptidecomprising amino acid sequence is 974-992 of SEQ ID NO: 1 operablylinked to a tolerogizing agent and a BoNT/B peptide comprising aminoacid sequence is 1058-1076 of SEQ ID NO: 1 operably linked to atolerogizing agent. In yet another aspect of this embodiment, atolerogizing composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-992 of SEQ ID NO: 1 operably linked to atolerogizing agent and a BoNT/B peptide comprising amino acid sequenceis 890-908 of SEQ ID NO: 1 operably linked to a tolerogizing agent.

In yet another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-984 ofSEQ ID NO: 1 operably linked to a tolerogizing agent and a BoNT/Bpeptide comprising amino acid sequence is 735-745 of SEQ ID NO: 1operably linked to a tolerogizing agent. In yet another aspect of thisembodiment, a tolerogizing composition comprises a BoNT/B peptidecomprising amino acid sequence is 974-984 of SEQ ID NO: 1 operablylinked to a tolerogizing agent and a BoNT/B peptide comprising aminoacid sequence is 1065-1075 of SEQ ID NO: 1 operably linked to atolerogizing agent. In yet another aspect of this embodiment, atolerogizing composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-984 of SEQ ID NO: 1 operably linked to atolerogizing agent and a BoNT/B peptide comprising amino acid sequenceis 895-905 of SEQ ID NO: 1 operably linked to a tolerogizing agent.

In still another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-992 ofSEQ ID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising amino acid sequence is 736-754 of SEQ ID NO: 1 operablylinked to a tolerogizing agent and a BoNT/B peptide comprising aminoacid sequence is 1058-1076 of SEQ ID NO: 1 operably linked to atolerogizing agent. In still another aspect of this embodiment, atolerogizing composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-992 of SEQ ID NO: 1 operably linked to atolerogizing agent, a BoNT/B peptide comprising amino acid sequence is736-754 of SEQ ID NO: 1 operably linked to a tolerogizing agent and aBoNT/B peptide comprising amino acid sequence is 890-908 of SEQ ID NO: 1operably linked to a tolerogizing agent.

In still another aspect of this embodiment, a tolerogizing compositioncomprises a BoNT/B peptide comprising amino acid sequence is 974-984 ofSEQ ID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising amino acid sequence is 735-745 of SEQ ID NO: 1 operablylinked to a tolerogizing agent and a BoNT/B peptide comprising aminoacid sequence is 1065-1075 of SEQ ID NO: 1 operably linked to atolerogizing agent. In still another aspect of this embodiment, atolerogizing composition comprises a BoNT/B peptide comprising aminoacid sequence is 974-984 of SEQ ID NO: 1 operably linked to atolerogizing agent, a BoNT/B peptide comprising amino acid sequence is735-745 of SEQ ID NO: 1 operably linked to a tolerogizing agent and aBoNT/B peptide comprising amino acid sequence is 895-905 of SEQ ID NO: 1operably linked to a tolerogizing agent.

In another embodiment, a tolerogizing composition comprises one or moredifferent BoNT/B peptides derived from a non-naturally occurring BoNT/B,each BoNT/B peptide operably linked to a tolerogizing agent. In aspectsof this embodiment, the non-naturally occurring BoNT/B is a conservativeBoNT/B variant or a non-conservative BoNT/B variant.

In aspects of this embodiment, a tolerogizing composition comprises oneor more different BoNT/B peptides derived from a conservative BoNT/Bvariant, two or more different BoNT/B peptides derived from aconservative BoNT/B variant, three or more different BoNT/B peptidesderived from a conservative BoNT/B variant, four or more differentBoNT/B peptides derived from a conservative BoNT/B variant, five or moredifferent BoNT/B peptides derived from a conservative BoNT/B variant,six or more different BoNT/B peptides derived from a conservative BoNT/Bvariant, seven or more different BoNT/B peptides derived from aconservative BoNT/B variant, eight or more different BoNT/B peptidesderived from a conservative BoNT/B variant, nine or more differentBoNT/B peptides derived from a conservative BoNT/B variant, ten or moredifferent BoNT/B peptides derived from a conservative BoNT/B variant, 15or more different BoNT/B peptides derived from a conservative BoNT/Bvariant or 20 or more different BoNT/B peptides derived from aconservative BoNT/B variant.

In aspects of this embodiment, a tolerogizing composition comprises oneor more BoNT/B peptides selected from the group consisting of a BoNT/Bpeptide comprising 1-4 conservative amino acid substitutions to aminoacids 610-628 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 736-754 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 conservative aminoacid substitutions to amino acids 820-838 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 862-880 of SEQ IDNO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 918-936 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 conservative aminoacid substitutions to amino acids 960-978 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 974-992 of SEQ IDNO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 1072-1090 of SEQ ID NO: 1 operably linkedto a tolerogizing agent, a BoNT/B peptide comprising 1-4 conservativeamino acid substitutions to amino acids 1254-1272 of SEQ ID NO: 1operably linked to a tolerogizing agent, and a BoNT/B peptide comprising1-4 conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 1 operably linked to a tolerogizing agent.

In another aspect of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides derived from a conservative BoNT/Band operably linked to a tolerogizing agent. In aspects of thisembodiment, a tolerogizing composition comprises one or more BoNT/Bpeptides selected from the group consisting of a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 778-796 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 conservative aminoacid substitutions to amino acids 932-950 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 974-992 of SEQ IDNO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids1058-1076 of SEQ ID NO: 1 operably linked to a tolerogizing agent, and aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 1 operably linked to a tolerogizingagent.

In further aspects of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides selected from the group consistingof a BoNT/B peptide comprising 1-4 conservative amino acid substitutionsto amino acids 616-626 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 735-745 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 conservative aminoacid substitutions to amino acids 778-789 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 867-877 of SEQ IDNO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids895-905 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 conservative amino acid substitutions toamino acids 929-939 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 conservative aminoacid substitutions to amino acids 1039-1049 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4conservative amino acid substitutions to amino acids 1065-1075 of SEQ IDNO: 1 operably linked to a tolerogizing agent or a BoNT/B peptidecomprising 1-4 conservative amino acid substitutions to amino acids1269-1281 of SEQ ID NO: 1 operably linked to a tolerogizing agent.

In further aspects of this embodiment, a tolerogizing compositioncomprises one or more different BoNT/B peptides derived from anon-conservative BoNT/B variant, two or more different BoNT/B peptidesderived from a non-conservative BoNT/B variant, three or more differentBoNT/B peptides derived from a non-conservative BoNT/B variant, four ormore different BoNT/B peptides derived from a non-conservative BoNT/Bvariant, five or more different BoNT/B peptides derived from anon-conservative BoNT/B variant, six or more different BoNT/B peptidesderived from a non-conservative BoNT/B variant, seven or more differentBoNT/B peptides derived from a non-conservative BoNT/B variant, eight ormore different BoNT/B peptides derived from a non-conservative BoNT/Bvariant, nine or more different BoNT/B peptides derived from anon-conservative BoNT/B variant, ten or more different BoNT/B peptidesderived from a non-conservative BoNT/B variant, 15 or more differentBoNT/B peptides derived from a non-conservative BoNT/B variant or 20 ormore different BoNT/B peptides derived from a non-conservative BoNT/Bvariant.

In another aspect of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides derived from a non-conservativeBoNT/B and operably linked to a tolerogizing agent. In further aspectsof this embodiment, a tolerogizing composition comprises one or moreBoNT/B peptides selected from the group consisting of a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids610-628 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 736-754 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 820-838 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 862-880 of SEQID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 918-936 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 960-978 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 1058-1076 of SEQ ID NO: 1 operably linked to atolerogizing agent, a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 1072-1090 of SEQ ID NO: 1operably linked to a tolerogizing agent, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 1254-1272 ofSEQ ID NO: 1 operably linked to a tolerogizing agent, and a BoNT/Bpeptide comprising 1-4 non-conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 1 operably linked to a tolerogizingagent.

In further aspects of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides selected from the group consistingof a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 736-754 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 778-796 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 974-992 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 1 operably linkedto a tolerogizing agent, and a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 1 operably linked to a tolerogizing agent.

In further other aspects of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides selected from the group consistingof a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 616-626 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 735-745 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 778-789 of SEQID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids867-877 of SEQ ID NO: 1 operably linked to a tolerogizing agent, aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 895-905 of SEQ ID NO: 1 operably linked to a tolerogizingagent, a BoNT/B peptide comprising 1-4 non-conservative amino acidsubstitutions to amino acids 929-939 of SEQ ID NO: 1 operably linked toa tolerogizing agent, a BoNT/B peptide comprising 1-4 non-conservativeamino acid substitutions to amino acids 974-984 of SEQ ID NO: 1 operablylinked to a tolerogizing agent, a BoNT/B peptide comprising 1-4non-conservative amino acid substitutions to amino acids 1039-1049 ofSEQ ID NO: 1 operably linked to a tolerogizing agent, a BoNT/B peptidecomprising 1-4 non-conservative amino acid substitutions to amino acids1065-1075 of SEQ ID NO: 1 operably linked to a tolerogizing agent or aBoNT/B peptide comprising 1-4 non-conservative amino acid substitutionsto amino acids 1269-1281 of SEQ ID NO: 1 operably linked to atolerogizing agent.

In still other aspects of this embodiment, a tolerogizing compositioncomprises one or more different BoNT/B peptides each peptide comprisinga different BoNT/B immunoreactive fragment, two or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment, three or more different BoNT/B peptides each peptidecomprising a different BoNT/B immunoreactive fragment, four or moredifferent BoNT/B peptides each peptide comprising a different BoNT/Bimmunoreactive fragment, five or more different BoNT/B peptides eachpeptide comprising a different BoNT/B immunoreactive fragment, six ormore different BoNT/B peptides each peptide comprising a differentBoNT/B immunoreactive fragment, seven or more different BoNT/B peptideseach peptide comprising a different BoNT/B immunoreactive fragment,eight or more different BoNT/B peptides each peptide comprising adifferent BoNT/B immunoreactive fragment, nine or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment, ten or more different BoNT/B peptides each peptide comprisinga different BoNT/B immunoreactive fragment, 15 or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment, 20 or more different BoNT/B peptides each peptide comprising adifferent BoNT/B immunoreactive fragment, 25 or more different BoNT/Bpeptides each peptide comprising a different BoNT/B immunoreactivefragment or 30 or more different BoNT/B peptides each peptide comprisinga different BoNT/B immunoreactive fragment.

In an aspect of this embodiment, a tolerogizing composition comprisesone or more BoNT/B peptides, each BoNT/B peptide operably linked to atolerogizing agent and each peptide comprising a different BoNT/Bimmunoreactive fragment capable of reducing an immunogenic response. Inother aspects of this embodiment, a tolerogizing composition comprisesone or more BoNT/B peptides, each peptide comprising a different BoNT/Bimmunoreactive fragment, each BoNT/B immunoreactive fragment comprisingat least six consecutive amino acids selected from the group consistingof amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ IDNO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQID NO: 1, amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 ofSEQ ID NO: 1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids862-880 of SEQ ID NO: 1, amino acids 862-880 of SEQ ID NO: 7, aminoacids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3,amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO:7, amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ IDNO: 3, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQID NO: 5, amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 ofSEQ ID NO: 3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, aminoacids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5,amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ IDNO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 ofSEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, amino acids1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 of SEQ ID NO: 3, aminoacids 1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1,amino acids 1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 1, amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 ofSEQ ID NO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides, each peptide comprising adifferent BoNT/B immunoreactive fragment, each BoNT/B immunoreactivefragment comprising at least six consecutive amino acids selected fromthe group consisting of amino acids 616-626 of SEQ ID NO: 1, amino acids735-745 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5, aminoacids 778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ ID NO:1, amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQ IDNO: 1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049 ofSEQ ID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids1039-1049 of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, aminoacids 1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5,amino acids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ IDNO: 1, amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 ofSEQ ID NO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

In still other aspects of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides, each peptide comprising adifferent BoNT/B immunoreactive fragment, each BoNT/B immunoreactivefragment comprising at least six non-consecutive amino acids selectedfrom the group consisting of amino acids 610-628 of SEQ ID NO: 1, aminoacids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5,amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO:5, amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ IDNO: 3, amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQID NO: 7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 ofSEQ ID NO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids890-908 of SEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, aminoacids 918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1,amino acids 932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO:1, amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ IDNO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 ofSEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, aminoacids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3,amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ IDNO: 7, amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 ofSEQ ID NO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3,amino acids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQID NO: 7.

In still other aspects of this embodiment, a tolerogizing compositioncomprises one or more BoNT/B peptides, each peptide comprising adifferent BoNT/B immunoreactive fragment, each BoNT/B immunoreactivefragment comprising at least six non-consecutive amino acids selectedfrom the group consisting of amino acids 616-626 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5,amino acids 778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:1, amino acids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ IDNO: 1, amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 ofSEQ ID NO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049of SEQ ID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids1039-1049 of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, aminoacids 1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5,amino acids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ IDNO: 1, amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 ofSEQ ID NO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

It is also envisioned that any and all combinations of BoNT/B peptidesdisclosed in the specification can be used in a tolerogizingcomposition, including, without limitation, a BoNT/B peptide derivedfrom a naturally occurring BoNT/B, such as, e.g., the BoNT/B of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11 or SEQ ID NO: 13, a BoNT/B isoform or a BoNT/B subtype; and aBoNT/B peptide derived from a non-naturally occurring BoNT/B, such as,e.g., a conservative BoNT/B variant, a non-conservative BoNT/B variantand a chimeric BoNT/B peptide. Thus, aspects of this embodiment, atolerogizing composition comprises, e.g., one or more BoNT/B peptidesderived from a naturally occurring BoNT/B and one or more BoNT/Bpeptides derived from a non-naturally occurring BoNT/B. In other aspectsof this embodiment, a tolerogizing composition comprises, e.g., one ormore BoNT/B peptides comprising one or more BoNT/B peptides derived fromSEQ ID NO: 1 and one or more BoNT/B peptides derived from a conservativeBoNT/B variant; one or more BoNT/B peptides of SEQ ID NO: 1 and one ormore BoNT/B peptides derived from a non-conservative variant; one ormore BoNT/B peptides derived from a conservative BoNT/B variant and oneor more BoNT/B peptides derived from a non-conservative BoNT/B variant;and one or more BoNT/B peptides derived from SEQ ID NO: 1, one or moreBoNT/B peptides derived from a conservative BoNT/B variant and one ormore BoNT/B peptides derived from a non-conservative BoNT/B variant.

In another embodiment, a tolerogizing composition comprises atolerogizing agent. In an aspect of this embodiment, a BoNT/B peptide isoperably linked to polyethylene glycol (PEG). In another aspect of thisembodiment, a BoNT/B peptide is operably linked tomonomethoxypolyethylene glycol (mPEG). In another aspect of thisembodiment, a BoNT/B peptide is operably linked to polyvinyl alcohol(PVA).

The present invention provides, in part, an immune response inducingcomposition comprising an adjuvant and a BoNT/B antigen. Such immuneresponse inducing compositions are useful for inducing specific immunityagainst one or more botulinum toxins such as, e.g., BoNT/B. Suchspecific immunity can protect an individual from intoxication producedby exposure to botulinum toxin. As used herein, the term “immuneresponse inducing composition” means a composition which, whenadministered to an individual, stimulates an immune response against anantigen. The term “immune response” refers to any response by the immunesystem of an individual to an immune response inducing composition orother immunogenic compound. Exemplary immune responses include, but notlimited to cellular as well as local and systemic humoral immunity, suchas CTL responses, including antigen-specific induction of CD8+ CTLs,helper T-cell responses, including T-cell proliferative responses andcytokine release, and B-cell responses including, e.g., an antibodyproducing response. The term “inducing an immune response” refers toadministration of an immune response inducing composition or otherimmunogenic compound or a nucleic acid encoding the immune responseinducing composition or other immunogenic compound, wherein an immuneresponse is affected, i.e., stimulated, initiated or induced. An immuneresponse inducing composition can be useful, for example, for preventingor ameliorating intoxication produced by unwanted exposure to botulinumtoxin. Administration of an immune response inducing composition hasbeen shown to effectively block the effect of protein toxins, see, e.g.,Behzod Z. Dolimbek & M. Zouhair Atassi, 13(5) J. PROT. CHEM. 490-493(1994); M. Zouhair Atassi et al., Antibody and T-Cell Recognition ofAlpha-Bungarotoxin and its Synthetic Loop-Peptides, 32(12) MOL. IMMUNOL.919-929 (1995); and Behzod Z. Dolimbek et al., Protection AgainstAlpha-Bungarotoxin Poisoning by Immunization with Synthetic ToxinPeptides, 33(7-8) MOL. IMMUNOL. 681-689 (1996).

Aspects of the present invention provide, in part, a BoNT/B antigen. Anantigen is a molecule that elicits an immune response and includes,without limitation, peptides, polysaccharides and conjugates of lipids,such as, e.g., lipoproteins and glycolipids. It is envisioned that anyof the BoNT/B peptides disclosed in the present specification can beuseful as a BoNT/B antigen, with the proviso that the BoNT/B peptideelicits an immunogenic response necessary to produce an anti-BoNTantibody that provides specific immunity against one or more botulinumtoxins, such as, e.g., BoNT/B. Non-limiting examples include a BoNT/Bpeptide derived from a naturally occurring BoNT/B, such as, e.g., theBoNT/B of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, a BoNT/B isoform or a BoNT/Bsubtype; and a BoNT/B peptide derived from a non-naturally occurringBoNT/B, such as, e.g., a conservative BoNT/B variant, a non-conservativeBoNT/B variant and a chimeric BoNT/B peptide. BoNT/B peptides disclosedin the present specification can be selected, e.g., depending onimmunological factors, such as potency of the peptide in inducing atolerogizing response, and technical factors, such as chemical synthesisyields. It is also understood that the two or more different BoNT/Bpeptides can be provided separately or as part of a compound moleculesuch as a chimeric BoNT/B peptide. An BoNT/B antigen disclosed in thepresent specification can be, e.g., prepared from natural sources,produced recombinantly, or synthesized chemically.

Thus, in an embodiment, a BoNT/B antigen has a length of at least 5amino acids and at most 60 amino acids. In an aspect of this embodiment,a BoNT/B antigen of SEQ ID NO: 1 has a length of at least 5 amino acidsand at most 60 amino acids. In other aspects of this embodiment, aBoNT/B antigen comprises an amino acid sequence selected from the groupconsisting of amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO: 5, aminoacids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ ID NO: 3,amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO:7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 of SEQ IDNO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 ofSEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1, amino acids932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO: 1, aminoacids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ ID NO: 7,amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO:3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ IDNO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 ofSEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, aminoacids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7,amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 of SEQ IDNO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 ofSEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, amino acids1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQ ID NO:7.

In other aspects of this embodiment, a BoNT/B antigen comprises an aminoacid sequence selected from the group consisting of amino acids 736-754of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO: 5, aminoacids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3,amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO:7, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ IDNO: 5, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1058-1076 ofSEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, aminoacids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3,amino acids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQID NO: 7.

In still other aspects of this embodiment, a BoNT/B antigen comprises anamino acid sequence selected from the group consisting of amino acids616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ IDNO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 ofSEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

In another embodiment, a BoNT/B antigen has a length of at least 5 aminoacids and at most 60 amino acids and is derived from a naturallyoccurring BoNT/B. In aspects of this embodiment, the naturally occurringBoNT/B is a BoNT/B isoform or a BoNT/B subtype. In other aspects of thisembodiment, a BoNT/B peptide is derived from the BoNT/B of SEQ ID NO: 1,the BoNT/B of SEQ ID NO: 3, the BoNT/B of SEQ ID NO: 5, the BoNT/B ofSEQ ID NO: 7, the BoNT/B of SEQ ID NO: 9, the BoNT/B of SEQ ID NO: 11 orthe BoNT/B of SEQ ID NO: 13.

In another embodiment, a BoNT/B antigen has a length of at least 5 aminoacids and at most 60 amino acids and is derived from a non-naturallyoccurring BoNT/B. In aspects of this embodiment, the naturally occurringBoNT/B is a conservative BoNT/B variant or a non-conservative BoNT/Bvariant.

In aspects of this embodiment, a BoNT/B antigen comprises, e.g., 1-4conservative amino acid substitutions to amino acids 610-628 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids 736-754of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 736-754 of SEQ ID NO: 5, 1-4 conservative amino acid substitutionsto amino acids 778-796 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 820-838 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 820-838 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 820-838of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 820-838 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 862-880 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 862-880 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 890-908 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 890-908of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 890-908 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 918-936 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 918-936 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 932-950 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 932-950 of SEQ IDNO: 5, 1-4 conservative amino acid substitutions to amino acids 960-978of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 960-978 of SEQ ID NO: 3, 1-4 conservative amino acid substitutionsto amino acids 960-978 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 974-992 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1030-1048 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 1030-1048 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 1030-1048 of SEQ ID NO: 7, 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids1058-1076 of SEQ ID NO: 3, 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 5, 1-4 conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 1072-1090 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1072-1090 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1072-1090 of SEQ ID NO: 7, 1-4 conservative amino acid substitutions toamino acids 1254-1272 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 1254-1272 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 1268-1291 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1268-1291 of SEQ ID NO: 5 or 1-4 conservative amino acid substitutionsto amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/B antigen comprises, e.g.,1-4 conservative amino acid substitutions to amino acids 736-754 of SEQID NO: 1, 1-4 conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 5, 1-4 conservative amino acid substitutions toamino acids 778-796 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 890-908 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 890-908of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 890-908 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 974-992 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 974-992of SEQ ID NO: 7, 1-4 conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 1058-1076 of SEQ ID NO: 5, 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1268-1291 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 5 or 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 7.

In still other aspects of this embodiment, a BoNT/B antigen comprises,e.g., 1-4 conservative amino acid substitutions to amino acids 616-626of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 735-745 of SEQ ID NO: 1, 1-4 conservative amino acid substitutionsto amino acids 735-745 of SEQ ID NO: 5, 1-4 conservative amino acidsubstitutions to amino acids 778-789 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 867-877 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 867-877 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids 895-905of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 895-905 of SEQ ID NO: 3, 1-4 conservative amino acid substitutionsto amino acids 929-939 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 974-984 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 974-984 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1039-1049 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1039-1049 of SEQ ID NO: 5, 1-4 conservative amino acidsubstitutions to amino acids 1039-1049 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 1065-1075 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1065-1075 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1065-1075 of SEQ ID NO: 5, 1-4 conservative amino acid substitutions toamino acids 1065-1075 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 1269-1281 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 1269-1281 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 1269-1281 of SEQ IDNO: 5 or 1-4 conservative amino acid substitutions to amino acids1269-1281 of SEQ ID NO: 7.

In aspects of this embodiment, a BoNT/B antigen comprises, e.g., 1-4non-conservative amino acid substitutions to amino acids 610-628 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 736-754 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 778-796 of SEQID NO: 5, 1-4 non-conservative amino acid substitutions to amino acids820-838 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 820-838 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 820-838 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 820-838 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids862-880 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 862-880 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 5, 1-4 non-conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 918-936 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 918-936 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 960-978 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 960-978 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids960-978 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 974-992 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 1030-1048 of SEQ ID NO: 3, 1-4 non-conservative aminoacid substitutions to amino acids 1030-1048 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 1030-1048 ofSEQ ID NO: 7, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 5, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1072-1090 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1072-1090 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1072-1090 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1254-1272 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1254-1272 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 5 or 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/B antigen comprises, e.g.,1-4 non-conservative amino acid substitutions to amino acids 736-754 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 736-754 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 778-796 of SEQID NO: 5, 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids974-992 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 1058-1076 of SEQ ID NO: 1, 1-4 non-conservative aminoacid substitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 5, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 5 or 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B antigen comprises,e.g., 1-4 non-conservative amino acid substitutions to amino acids616-626 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 735-745 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 735-745 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 778-789 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids867-877 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 867-877 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 895-905 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 895-905 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids929-939 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 974-984 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 974-984 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids1039-1049 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 1039-1049 of SEQ ID NO: 5, 1-4 non-conservative aminoacid substitutions to amino acids 1039-1049 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 1065-1075 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 1065-1075 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 7, 1-4 non-conservative amino acid substitutions to aminoacids 1269-1281 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1269-1281 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1269-1281 ofSEQ ID NO: 5 or 1-4 non-conservative amino acid substitutions to aminoacids 1269-1281 of SEQ ID NO: 7.

In another embodiment, an immunoreactive fragment of a BoNT/B antigenhas a length of at least five amino acids and at most 60 amino acids. Inaspects of this embodiment, an immunoreactive fragment of a BoNT/Bantigen comprises, e.g., at least 6 consecutive amino acids, at least 7consecutive amino acids, at least 8 consecutive amino acids, at least 9consecutive amino acids, at least 10 consecutive amino acids, at least12 consecutive amino acids, at least 15 consecutive amino acids, atleast 18 consecutive amino acids or at least 20 consecutive amino acids.In other aspects of this embodiment, an immunoreactive fragment of aBoNT/B antigen comprises, e.g., at most 6 consecutive amino acids, atmost 7 consecutive amino acids, at most 8 consecutive amino acids, atmost 9 consecutive amino acids, at most 10 consecutive amino acids, atmost 12 consecutive amino acids, at most 15 consecutive amino acids, atmost 18 consecutive amino acids or at most 20 consecutive amino acids.

In still other aspects of this embodiment, an immunoreactive fragment ofa BoNT/B antigen comprises, e.g., at least 6 non-consecutive aminoacids, at least 7 non-consecutive amino acids, at least 8non-consecutive amino acids, at least 9 non-consecutive amino acids, atleast 10 non-consecutive amino acids, at least 12 non-consecutive aminoacids, at least 15 non-consecutive amino acids, at least 18non-consecutive amino acids or at least 20 non-consecutive amino acids.In still other aspects of this embodiment, an immunoreactive fragment ofa BoNT/B antigen comprises, e.g., at most 6 non-consecutive amino acids,at most 7 non-consecutive amino acids, at most 8 non-consecutive aminoacids, at most 9 non-consecutive amino acids, at most 10 non-consecutiveamino acids, at most 12 non-consecutive amino acids, at most 15non-consecutive amino acids, at most 18 non-consecutive amino acids orat most 20 non-consecutive amino acids.

In still other aspects of this embodiment, an immunoreactive fragment ofa BoNT/B antigen has from five to sixty amino acids, from five to fiftyamino acids, from eight to fifty amino acids, from ten to fifty aminoacids, from five to twenty amino acids, from eight to twenty aminoacids, from ten to twenty amino acids, from twelve to twenty amino acidsor from fifteen to twenty amino acids.

In another embodiment, a BoNT/B antigen comprises an immunogenic BoNT/Bfragment. In an aspect of this embodiment, an immunogenic BoNT/Bfragment comprises at least six consecutive amino acids of a BoNT/Bantigen. In other aspects of this embodiment, an immunogenic BoNT/Bfragment comprises at least six consecutive amino acids from, e.g.,amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO:1, amino acids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ IDNO: 1, amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQID NO: 1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838 ofSEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880of SEQ ID NO: 1, amino acids 862-880 of SEQ ID NO: 7, amino acids890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3, aminoacids 890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 7,amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ ID NO:3, amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ IDNO: 5, amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQID NO: 3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992 ofSEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, aminoacids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1,amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ IDNO: 5, amino acids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 ofSEQ ID NO: 1, amino acids 1072-1090 of SEQ ID NO: 3, amino acids1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1, aminoacids 1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1,amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six consecutive amino acids from, e.g., amino acids616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ IDNO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 ofSEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

In another aspect of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six non-consecutive amino acids of a BoNT/B antigen.In other aspects of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six non-consecutive amino acids from, e.g., aminoacids 610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 1,amino acids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO:1, amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQ IDNO: 1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838 of SEQID NO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880 ofSEQ ID NO: 1, amino acids 862-880 of SEQ ID NO: 7, amino acids 890-908of SEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3, amino acids890-908 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 7, aminoacids 918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ ID NO: 3,amino acids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ ID NO:5, amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQ IDNO: 3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992 of SEQID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 ofSEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, aminoacids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1,amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ IDNO: 5, amino acids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 ofSEQ ID NO: 1, amino acids 1072-1090 of SEQ ID NO: 3, amino acids1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1, aminoacids 1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1,amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six non-consecutive amino acids from, e.g., aminoacids 616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1,amino acids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO:1, amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ IDNO: 7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQID NO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 ofSEQ ID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984of SEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

In is envisioned that an immune response inducing composition canoptionally comprises one or more carriers to enhance the immunogenicityof an antigen, a hapten, or any other antigenic compound that isimmunogenic, non-immunogenic, or weakly immunogenic when not associatedwith the carrier. As is well known in the art, a non-antigenic or weaklyantigenic antigen can be made antigenic by coupling the antigen to acarrier, such as, e.g., keyhole limpet hemacyanin (KLH), ovalbumin(OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsininhibitor (STI) or multiple attachment peptide (MAP) technology. Variousother carrier and methods for coupling an antigen to a carrier are wellknown in the art, see, e.g., Harlow and Lane, supra, 1998a; Harlow andLane, supra, 1998b; and David W. Waggoner, Jr. et al.,Immunogenicity-enhancing carriers and compositions thereof and methodsof using the same, U.S. Patent Publication No. 20040057958 (Mar. 25,2004). An epitope can also be generated by expressing the epitope as afusion protein, for example, fused to glutathione S transferase, polyHisor the like. Methods for expressing polypeptide fusions are well knownto those skilled in the art as described, for example, in Ausubel etal., Current Protocols in Molecular Biology (Supplement 47), John Wiley& Sons, New York (1999).

In is envisioned that an immune response inducing composition alsooptionally comprises one or more adjuvants. As used herein, the term“adjuvant” when used in reference to an immune response inducingcomposition means any substance or mixture of substances that increasesor diversifies the immune response to an antigenic compound. An immuneresponse inducing adjuvant can, for example, serve to reduce the numberof immunizations or the amount of antigen required for protectiveimmunization. The use of immune response inducing adjuvants in an immuneresponse inducing composition is well known. The main objective of theseadjuvants is to allow an increase in the immune response. Theseadjuvants are diverse in nature. They may, e.g., consist of liposomes,oily phases, including, without limitation, the Freund type ofadjuvants, such as, e.g., Freund's complete adjuvant (FCA); Freund'sincomplete adjuvant (FIA); sapogenin glycosides, such as, e.g.,saponins; carbopol; N-acetylmuramyl-L-alanyl-D-isoglutamine (commonlyknown as muramyl dipeptide or “MDP”); and lipopolysaccharide (LPS). Suchadjuvants are generally used in the form of an emulsion with an aqueousphase, or, more commonly, may consist of water-insoluble inorganicsalts. These inorganic salts may consist, for example, of aluminumhydroxide, zinc sulfate, colloidal iron hydroxide, calcium phosphate orcalcium chloride. Aluminum hydroxide (Al(OH)₃) is a commonly usedadjuvant. Currently, the only FDA-approved adjuvant for use in humans isaluminum salts (Alum) which are used to “depot” antigens byprecipitation of the antigens. Adjuvants provided above are merelyexemplary. In fact, any immune response inducing adjuvant may be used inan immune response inducing composition disclosed in the presentspecification as long as the adjuvant satisfies the requisitecharacteristics that are necessary for practicing the present invention.

As indicated above, the carrier of the compositions of the presentinvention itself may act as an adjuvant. Specific adjuvants and methodsof making and using are described in, e.g., Gupta et al. Vaccine, 11:993-306, 1993; Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press,Inc., Boca Raton, Fla., 1987; and David W. Waggoner, Jr. et al.,Immunogenicity-Enhancing Carriers and Compositions Thereof and Methodsof Using the Same, U.S. Patent Publication No. 20040057958 (Mar. 25,2004). Additional adjuvants include any compound described in Chapter 7(pp 141-227) of “Vaccine Design, The Subunit and Adjuvant Approach”(eds. Powell, M. F. and Newman, M. J.) Pharmaceutical Biotechnology,Volume 6, Plenum Press (New York). Examples from this compendium includeMuramyl Dipeptide (MDP) and Montanide 720. Molecules such as PolyInosine:Cytosine (Poly I:C) or plasmid DNA containing CpG motifs canalso be administered as adjuvants in combination with antigensencapsulated in microparticles. In another example, the adjuvant is anagent that facilitates entry of the antigenic compound into thecytoplasm of a cell such as listeriolysin, streptolysin or a mixturethereof.

In an embodiment, an immune response inducing composition comprises aBoNT/B antigen disclosed in the present specification operably linked toa carrier. In another embodiment, an immune response inducingcomposition can comprise a plurality of different BoNT/B antigensdisclosed in the present specification each BoNT/B antigen operablylinked to a carrier. Thus, in aspects of this embodiment, an immuneresponse inducing composition comprises one or more different BoNT/Bantigens each BoNT/B antigen operably linked to a carrier, two or moredifferent BoNT/B antigens each BoNT/B peptide operably linked to acarrier, three or more different BoNT/B antigens each BoNT/B antigenoperably linked to a carrier, four or more different BoNT/B antigenseach BoNT/B antigen operably linked to a carrier, five or more differentBoNT/B antigens, each BoNT/B antigen operably linked to a carrier, sixor more different BoNT/B antigens each BoNT/B antigen operably linked toa carrier, seven or more different BoNT/B antigens each BoNT/B antigenoperably linked to a carrier, eight or more different BoNT/B antigenseach BoNT/B antigen operably linked to a carrier, nine or more differentBoNT/B antigens each BoNT/B antigen operably linked to a carrier, ten ormore different BoNT/B antigens each BoNT/B antigen operably linked to acarrier, 15 or more different BoNT/B antigens each BoNT/B antigenoperably linked to a carrier or 20 or more different BoNT/B antigenseach BoNT/B antigen operably linked to a carrier.

In another embodiment, an immune response inducing composition comprisesone or more different BoNT/B antigens derived from the BoNT/B of SEQ IDNO: 1, the BoNT/B of SEQ ID NO: 3, the BoNT/B of SEQ ID NO: 5, theBoNT/B of SEQ ID NO: 7, the BoNT/B of SEQ ID NO: 9, the BoNT/B of SEQ IDNO: 11 or the BoNT/B of SEQ ID NO: 13.

In aspects of this embodiment, an immune response inducing compositioncomprises one or more different BoNT/B antigens comprising an amino acidsequence selected from the group consisting of amino acids 610-628 ofSEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 1, aminoacids 820-838 of SEQ ID NO: 3, amino acids 820-838 of SEQ ID NO: 5,amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880 of SEQ ID NO:1, amino acids 862-880 of SEQ ID NO: 7, amino acids 890-908 of SEQ IDNO: 1, amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQID NO: 5, amino acids 890-908 of SEQ ID NO: 7, amino acids 918-936 ofSEQ ID NO: 1, amino acids 918-936 of SEQ ID NO: 3, amino acids 932-950of SEQ ID NO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQ ID NO: 3, aminoacids 960-978 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1,amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO:7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, aminoacids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 of SEQ ID NO: 1,amino acids 1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 of SEQ IDNO: 7, amino acids 1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 ofSEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1, amino acids1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5 andamino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, an immune response inducingcomposition comprises one or more amino acid sequences selected from thegroup consisting of amino acids 616-626 of SEQ ID NO: 1, amino acids735-745 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5, aminoacids 778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ ID NO:1, amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQ IDNO: 1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049 ofSEQ ID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids1039-1049 of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, aminoacids 1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5,amino acids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ IDNO: 1, amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 ofSEQ ID NO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

In another aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen operably linked to a carrier. Inother aspects of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen operably linked to a carrier, theBoNT/B antigen comprising amino acid sequence selected from the groupconsisting of amino acids 610-628 of SEQ ID NO: 1, amino acids 736-754of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO: 5, aminoacids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ ID NO: 3,amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO:7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 of SEQ IDNO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908 of SEQID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908 ofSEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, amino acids 918-936of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1, amino acids932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO: 1, aminoacids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ ID NO: 7,amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO:3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ IDNO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 ofSEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, aminoacids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7,amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 of SEQ IDNO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 ofSEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, amino acids1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQ ID NO:7.

In another aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen operably linked to a carrier, theBoNT/B antigen comprising amino acid sequence selected from the groupconsisting of amino acids 616-626 of SEQ ID NO: 1, amino acids 735-745of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5, amino acids778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 1, aminoacids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ ID NO: 1,amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQ ID NO:1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 of SEQ IDNO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049 of SEQID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids 1039-1049of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, amino acids1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5, aminoacids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ ID NO: 1,amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 of SEQ IDNO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

In yet another aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-992 of SEQ ID NO: 1 operably linked to a carrier and a BoNT/Bantigen comprising amino acid sequence is 736-754 of SEQ ID NO: 1operably linked to a carrier. In yet another aspect of this embodiment,an immune response inducing composition comprises a BoNT/B antigencomprising amino acid sequence is 974-992 of SEQ ID NO: 1 operablylinked to a carrier and a BoNT/B antigen comprising amino acid sequenceis 1058-1076 of SEQ ID NO: 1 operably linked to a carrier. In yetanother aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-992 of SEQ ID NO: 1 operably linked to a carrier and a BoNT/Bantigen comprising amino acid sequence is 890-908 of SEQ ID NO: 1operably linked to a carrier.

In yet another aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-984 of SEQ ID NO: 1 operably linked to a carrier and a BoNT/Bantigen comprising amino acid sequence is 735-745 of SEQ ID NO: 1operably linked to a carrier. In yet another aspect of this embodiment,an immune response inducing composition comprises a BoNT/B antigencomprising amino acid sequence is 974-984 of SEQ ID NO: 1 operablylinked to a carrier and a BoNT/B antigen comprising amino acid sequenceis 1065-1075 of SEQ ID NO: 1 operably linked to a carrier. In yetanother aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-984 of SEQ ID NO: 1 operably linked to a carrier and a BoNT/Bantigen comprising amino acid sequence is 895-905 of SEQ ID NO: 1operably linked to a carrier.

In still another aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-992 of SEQ ID NO: 1 operably linked to a carrier, a BoNT/B antigencomprising amino acid sequence is 736-754 of SEQ ID NO: 1 operablylinked to a carrier and a BoNT/B antigen comprising amino acid sequenceis 1058-1076 of SEQ ID NO: 1 operably linked to a carrier. In stillanother aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-992 of SEQ ID NO: 1 operably linked to a carrier, a BoNT/B antigencomprising amino acid sequence is 736-754 of SEQ ID NO: 1 operablylinked to a carrier and a BoNT/B antigen comprising amino acid sequenceis 890-908 of SEQ ID NO: 1 operably linked to a carrier.

In still another aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-984 of SEQ ID NO: 1 operably linked to a carrier, a BoNT/B antigencomprising amino acid sequence is 735-745 of SEQ ID NO: 1 operablylinked to a carrier and a BoNT/B antigen comprising amino acid sequenceis 1065-1075 of SEQ ID NO: 1 operably linked to a carrier. In stillanother aspect of this embodiment, an immune response inducingcomposition comprises a BoNT/B antigen comprising amino acid sequence is974-984 of SEQ ID NO: 1 operably linked to a carrier, a BoNT/B antigencomprising amino acid sequence is 735-745 of SEQ ID NO: 1 operablylinked to a carrier and a BoNT/B antigen comprising amino acid sequenceis 895-905 of SEQ ID NO: 1 operably linked to a carrier.

In another embodiment, an immune response inducing composition comprisesone or more different BoNT/B antigens derived from a non-naturallyoccurring BoNT/B, each BoNT/B peptide operably linked to a carrier. Inaspects of this embodiment, the non-naturally occurring BoNT/B is aconservative BoNT/B variant or a non-conservative BoNT/B variant.

In aspects of this embodiment, an immune response inducing compositioncomprises one or more different BoNT/B antigens derived from aconservative BoNT/B variant, two or more different BoNT/B antigensderived from a conservative BoNT/B variant, three or more differentBoNT/B antigens derived from a conservative BoNT/B variant, four or moredifferent BoNT/B antigens derived from a conservative BoNT/B variant,five or more different BoNT/B antigens derived from a conservativeBoNT/B variant, six or more different BoNT/B antigens derived from aconservative BoNT/B variant, seven or more different BoNT/B antigensderived from a conservative BoNT/B variant, eight or more differentBoNT/B antigens derived from a conservative BoNT/B variant, nine or moredifferent BoNT/B antigens derived from a conservative BoNT/B variant,ten or more different BoNT/B antigens derived from a conservative BoNT/Bvariant, 15 or more different BoNT/B antigens derived from aconservative BoNT/B variant or 20 or more different BoNT/B antigensderived from a conservative BoNT/B variant.

In aspects of this embodiment, an immune response inducing compositioncomprises one or more BoNT/B antigens selected from the group consistingof a BoNT/B antigen comprising 1-4 conservative amino acid substitutionsto amino acids 610-628 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 736-754 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 778-796 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 820-838 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 862-880 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 890-908 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 918-936 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 932-950 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 960-978 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 974-992 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1030-1048 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1072-1090 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1254-1272 of SEQ ID NO: 1 operably linked to a carrier, anda BoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 1 operably linked to a carrier.

In aspects of this embodiment, an immune response inducing compositioncomprises one or more BoNT/B antigens selected from the group consistingof a BoNT/B antigen comprising 1-4 conservative amino acid substitutionsto amino acids 736-754 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 778-796 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 890-908 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 932-950 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 974-992 of SEQ ID NO: 1 operably linked to a carrier, aBoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 1 operably linked to a carrier, anda BoNT/B antigen comprising 1-4 conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 1 operably linked to a carrier.

In further aspects of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens selected from thegroup consisting of a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 616-626 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 735-745 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 778-789 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 867-877 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 895-905 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 929-939 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 974-984 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 1039-1049 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 conservative aminoacid substitutions to amino acids 1065-1075 of SEQ ID NO: 1 operablylinked to a carrier or a BoNT/B antigen comprising 1-4 conservativeamino acid substitutions to amino acids 1269-1281 of SEQ ID NO: 1operably linked to a carrier.

In further aspects of this embodiment, an immune response inducingcomposition comprises one or more different BoNT/B antigens derived froma non-conservative BoNT/B variant, two or more different BoNT/B antigensderived from a non-conservative BoNT/B variant, three or more differentBoNT/B antigens derived from a non-conservative BoNT/B variant, four ormore different BoNT/B antigens derived from a non-conservative BoNT/Bvariant, five or more different BoNT/B antigens derived from anon-conservative BoNT/B variant, six or more different BoNT/B antigensderived from a non-conservative BoNT/B variant, seven or more differentBoNT/B antigens derived from a non-conservative BoNT/B variant, eight ormore different BoNT/B antigens derived from a non-conservative BoNT/Bvariant, nine or more different BoNT/B antigens derived from anon-conservative BoNT/B variant, ten or more different BoNT/B antigensderived from a non-conservative BoNT/B variant, 15 or more differentBoNT/B antigens derived from a non-conservative BoNT/B variant or 20 ormore different BoNT/B antigens derived from a non-conservative BoNT/Bvariant.

In further aspects of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens selected from thegroup consisting of a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 610-628 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 736-754 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 778-796 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 820-838 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 862-880 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 918-936 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 932-950 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 960-978 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 1030-1048 of SEQ ID NO: 1operably linked to a carrier, a BoNT/B antigen comprising 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 1 operably linked to a carrier, a BoNT/B antigen comprising1-4 non-conservative amino acid substitutions to amino acids 1072-1090of SEQ ID NO: 1 operably linked to a carrier, a BoNT/B antigencomprising 1-4 non-conservative amino acid substitutions to amino acids1254-1272 of SEQ ID NO: 1 operably linked to a carrier, and a BoNT/Bantigen comprising 1-4 non-conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 1 operably linked to a carrier.

In further aspects of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens selected from thegroup consisting of a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 736-754 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 778-796 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 932-950 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 974-992 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 1058-1076 of SEQ ID NO: 1operably linked to a carrier, and a BoNT/B antigen comprising 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 1 operably linked to a carrier.

In further other aspects of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens selected from thegroup consisting of a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 616-626 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 735-745 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 778-789 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 867-877 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 895-905 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 929-939 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 974-984 of SEQ ID NO: 1 operablylinked to a carrier, a BoNT/B antigen comprising 1-4 non-conservativeamino acid substitutions to amino acids 1039-1049 of SEQ ID NO: 1operably linked to a carrier, a BoNT/B antigen comprising 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 1 operably linked to a carrier or a BoNT/B antigen comprising1-4 non-conservative amino acid substitutions to amino acids 1269-1281of SEQ ID NO: 1 operably linked to a carrier.

In still other aspects of this embodiment, an immune response inducingcomposition comprises one or more different BoNT/B antigens each peptidecomprising a different BoNT/B immunoreactive fragment, two or moredifferent BoNT/B antigens each antigen comprising a different BoNT/Bimmunoreactive fragment, three or more different BoNT/B antigens eachantigen comprising a different BoNT/B immunoreactive fragment, four ormore different BoNT/B antigens each antigen comprising a differentBoNT/B immunoreactive fragment, five or more different BoNT/B antigenseach antigen comprising a different BoNT/B immunoreactive fragment, sixor more different BoNT/B antigens each antigen comprising a differentBoNT/B immunoreactive fragment, seven or more different BoNT/B antigenseach antigen comprising a different BoNT/B immunoreactive fragment,eight or more different BoNT/B antigens each antigen comprising adifferent BoNT/B immunoreactive fragment, nine or more different BoNT/Bantigens each antigen comprising a different BoNT/B immunoreactivefragment, ten or more different BoNT/B antigens each antigen comprisinga different BoNT/B immunoreactive fragment, 15 or more different BoNT/Bantigens each antigen comprising a different BoNT/B immunoreactivefragment, 20 or more different BoNT/B antigens each antigen comprising adifferent BoNT/B immunoreactive fragment, 25 or more different BoNT/Bantigens each antigen comprising a different BoNT/B immunoreactivefragment or 30 or more different BoNT/B antigens each antigen comprisinga different BoNT/B immunoreactive fragment.

In an aspect of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six consecutive amino acids of a BoNT/B antigen. Inother aspects of this embodiment, an immunogenic BoNT/B fragmentcomprises at least six consecutive amino acids from, e.g., amino acids610-628 of SEQ ID NO: 1

In an aspect of this embodiment, an immune response inducing compositioncomprises one or more BoNT/B antigens, each antigen comprising adifferent BoNT/B immunoreactive fragment. In another aspect of thisembodiment, an immune response inducing composition comprises one ormore BoNT/B antigens, each BoNT/B antigen comprising a different BoNT/Bimmunoreactive fragment, each BoNT/B immunoreactive fragment comprisingat least six consecutive amino acids of a BoNT/B antigen. In aspects ofthis embodiment, an immune response inducing composition comprises oneor more BoNT/B antigens, each BoNT/B antigen comprising a differentBoNT/B immunoreactive fragment, each BoNT/B immunoreactive fragmentcomprises at least six consecutive amino acids from, e.g., amino acids610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 1, aminoacids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1,amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO:1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838 of SEQ IDNO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880 of SEQID NO: 1, amino acids 862-880 of SEQ ID NO: 7, amino acids 890-908 ofSEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 7, amino acids918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ ID NO: 3, aminoacids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ ID NO: 5,amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQ ID NO:3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992 of SEQ IDNO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, aminoacids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,amino acids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 of SEQ IDNO: 1, amino acids 1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 ofSEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1, amino acids1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1, aminoacids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5and amino acids 1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens, each BoNT/B antigencomprising a different BoNT/B immunoreactive fragment, each BoNT/Bimmunoreactive fragment comprises at least six consecutive amino acidsfrom, e.g., amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 ofSEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 1, amino acids890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5, aminoacids 890-908 of SEQ ID NO: 7, amino acids 932-950 of SEQ ID NO: 1,amino acids 932-950 of SEQ ID NO: 5, amino acids 974-992 of SEQ ID NO:1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ IDNO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 ofSEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids1058-1076 of SEQ ID NO: 7, amino acids 1268-1291 of SEQ ID NO: 1, aminoacids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5and amino acids 1268-1291 of SEQ ID NO: 7.

In another aspect of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens, each BoNT/B antigencomprising a different BoNT/B immunoreactive fragment, each BoNT/Bimmunoreactive fragment comprising at least six non-consecutive aminoacids of a BoNT/B peptide. In aspects of this embodiment, an immuneresponse inducing composition comprises one or more BoNT/B peptides,each BoNT/B peptide comprising a different BoNT/B immunoreactivefragment, each BoNT/B immunoreactive fragment comprises at least sixnon-consecutive amino acids from, e.g., amino acids 610-628 of SEQ IDNO: 1, amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQID NO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 ofSEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838of SEQ ID NO: 3, amino acids 820-838 of SEQ ID NO: 5, amino acids820-838 of SEQ ID NO: 7, amino acids 862-880 of SEQ ID NO: 1, aminoacids 862-880 of SEQ ID NO: 7, amino acids 890-908 of SEQ ID NO: 1,amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO:5, amino acids 890-908 of SEQ ID NO: 7, amino acids 918-936 of SEQ IDNO: 1, amino acids 918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQID NO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids 960-978 ofSEQ ID NO: 1, amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, aminoacids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3,amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ IDNO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 ofSEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 of SEQ ID NO: 1, aminoacids 1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 of SEQ ID NO: 7,amino acids 1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 of SEQ IDNO: 3, amino acids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 ofSEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5 and amino acids1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, an immune response inducingcomposition comprises one or more BoNT/B antigens, each BoNT/B antigencomprising a different BoNT/B immunoreactive fragment, each BoNT/Bimmunoreactive fragment comprises at least six non-consecutive aminoacids from, e.g., amino acids 736-754 of SEQ ID NO: 1, amino acids736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1, aminoacids 778-796 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 1,amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO:5, amino acids 890-908 of SEQ ID NO: 7, amino acids 932-950 of SEQ IDNO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids 974-992 of SEQID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 ofSEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, aminoacids 1058-1076 of SEQ ID NO: 7, amino acids 1268-1291 of SEQ ID NO: 1,amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

It is also envisioned that any and all combinations of BoNT/B antigensdisclosed in the specification, including, without limitation, a BoNT/Bantigen derived from a naturally occurring BoNT/B, such as, e.g., theBoNT/B of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, a BoNT/B isoform or a BoNT/Bsubtype; and a BoNT/B peptide derived from a non-naturally occurringBoNT/B, such as, e.g., a conservative BoNT/B variant, a non-conservativeBoNT/B variant and a chimeric BoNT/B peptide. Thus, aspects of thisembodiment, an immune response inducing composition comprises, e.g., oneor more BoNT/B antigens derived from a naturally occurring BoNT/B andone or more BoNT/B antigens derived from a non-naturally occurringBoNT/B. In other aspects of this embodiment, an immune response inducingcomposition comprises, e.g., one or more BoNT/B antigens comprising oneor more BoNT/B antigens derived from SEQ ID NO: 1 and one or more BoNT/Bantigens derived from a conservative BoNT/B variant; one or more BoNT/Bpeptides of SEQ ID NO: 1 and one or more BoNT/B antigens derived from anon-conservative variant; one or more BoNT/B antigens derived from aconservative BoNT/B variant and one or more BoNT/B antigens derived froma non-conservative BoNT/B variant; and one or more BoNT/B antigensderived from SEQ ID NO: 1, one or more BoNT/B antigens derived from aconservative BoNT/B variant and one or more BoNT/B antigens derived froma non-conservative BoNT/B variant.

In another embodiment, an immune response inducing composition comprisesa carrier. In an aspect of this embodiment, a BoNT/B antigen is operablylinked to cholera enterotoxin A2. In another aspect of this embodiment,a BoNT/B antigen is operably linked to a peptide adjuvant.

In an embodiment, an immune response inducing composition comprising aBoNT/B antigen disclosed in the present specification operably linked toa carrier further comprises an adjuvant. In an aspect of thisembodiment, an adjuvant comprises a water-insoluble inorganic salt. Inanother aspect of this embodiment, an adjuvant comprises an aluminumsalt. In other aspects of this embodiment, an adjuvant comprises awater-insoluble inorganic salt selected from the group consisting ofaluminum hydroxide, zinc sulfate, colloidal iron hydroxide, calciumphosphate and calcium chloride. In yet another aspect of thisembodiment, an adjuvant comprises listeriolysin, streptolysin or anadmixture thereof. Patients treated with a BoNT therapy can developimmunoresistance to the treatment, thereby reducing or eliminating thebeneficial effect of the BoNT therapy. Therefore, methods that determinewhether a patient is mounting an immune response against a BoNT therapyare of major importance. These assays would allow the immunoresponsivestate of the patient to be evaluated periodically during the course of aBoNT therapy. By knowing the predisposition of an individual 1) thepotential value of a specific BoNT treatment can be determined prior toits administration to a patient; and 2) the possible benefit fromcontinued BoNT therapy can be assessed and any possible adjustments to atreatment determined. Therefore, these assays present a major benefit interms of providing better patient care and reducing health care costs.The BoNT/B peptides disclosed in the present specification are useful inmethods of determining immunoresistance to BoNT therapy in anindividual. For example, these peptides each contain one or moreepitopes recognized by anti-BoNT/B antibodies contained in antisera fromanimals immunized with BoNT/B, and thus can serve as binding substratesfor anti-BoNT antibodies.

Thus, the present invention provides, in part, a method of determiningimmunoresistance to botulinum toxin therapy in an individual bydetermining the presence or absence in the individual of anti-BoNTantibodies immunoreactive with a BoNT/B peptide disclosed in the presentspecification, where the presence of anti-BoNT antibodies immunoreactivewith the a BoNT/B peptide indicates immunoresistance to BoNT/B therapy.

The present invention also provides, in part, a method of determiningimmunoresistance to BoNT therapy in an individual, the method comprisingthe steps of combining a BoNT/B peptide and test sample and detectingthe amount of complexes formed by the BoNT/B peptide and an anti-BoNTantibody, where the presence of the antibody-peptide complex indicatesimmunoresistance to a BoNT therapy.

The present invention also provides, in part, a method of determiningimmunoresistance to BoNT therapy in an individual, the method comprisingthe steps of combining a BoNT/B peptide and a test sample, detecting theamount of complexes formed by the BoNT/B peptide and anti-BoNT antibodyand correlating the amount of the antibody-peptide complexes formed fromthe test sample relative to the amount of complexes formed by the BoNT/Bpeptide and the anti-BoNT antibody from a control sample.

It is further is understood that a methods of determiningimmunoresistance to BoNT therapy in an individual can be used to predictthe likelihood of the individual developing immunoresistance or toconfirm that the presence of anti-BoNT antibodies are a cause underlyingimmunoresistance to botulinum toxin therapy.

Aspects of the present invention provide, in part, determiningimmunoresistance to BoNT therapy in an individual. As used herein, theterm “immunoresistance,” when used in reference to botulinum toxintherapy, means an individual that does not fully respond to a botulinumtoxin therapy, or shows a reduced beneficial effect of botulinum toxintherapy resulting from the presence in the individual of anti-BoNTantibodies that bind to a botulinum toxin. Non-limiting examples of abotulinum toxin immunoresistance include, e.g., a BoNT/Aimmunoresistance, a BoNT/B immunoresistance, a BoNT/C1 immunoresistance,a BoNT/D immunoresistance, a BoNT/E immunoresistance, a BoNT/Fimmunoresistance and a BoNT/G immunoresistance. A non-limiting exampleof reduced efficacy would be the presence in an individual of at leastone neutralizing anti-BoNT/A antibody that binds to a BoNT/B toxin in amanner that reduces or prevents the specificity or activity of thetoxin. Another non-limiting example of reduced efficacy would be thepresence in an individual of at least one neutralizing anti-BoNT/Bantibody that binds to a BoNT/B toxin in a manner that reduces orprevents the specificity or activity of the toxin. As used herein, theterm “botulinum toxin therapy” is synonymous with “BoNT therapy” andmeans a treatment, remedy, cure, healing, rehabilitation or any othermeans of counteracting something undesirable in a mammal requiringneuromodulation using a botulinum toxin or administering to a mammal oneor more controlled doses of a medication, preparation or mixture of abotulinum toxin that has medicinal, therapeutic, curative, cosmetic,remedial or any other beneficial effect. Non-limiting examples of abotulinum toxin therapy include, e.g., a BoNT/A therapy, a BoNT/Btherapy, a BoNT/C1 therapy, a BoNT/D therapy, a BoNT/E therapy, a BoNT/Ftherapy and a BoNT/G therapy. The term botulinum toxin therapyencompasses, without limitation, the use of any naturally occurring ormodified or engineered form of a botulinum toxin or a domain or fragmentthereof, in any formulation, combined with any carrier or activeingredient and administered by any route of administration. Well-knownbotulinum toxin therapies include, without limitation, a BoNT/A therapy,such as, e.g., a BOTOX® therapy, a Dysport®/Reloxin® therapy, aLinurase® therapy, a Neuronox® therapy, a BTX-A therapy, and a Xeomin®therapy; and a BoNT/B therapy, such as, e.g., a MyoBloc™/NeuroBloc™therapy. Appropriate therapeutic and cosmetic uses of a botulinum toxintherapy are known in the art. As used herein, the term “individual,”when used in reference to botulinum toxin therapy, means any organismcapable of raising anti-BoNT antibodies against a BoNT toxin, including,but not limited to, birds and mammals, including mice, rats, goats,sheep, horses, donkeys, cows, primates and humans.

Aspects of the present invention provide, in part, a BoNT/B peptide. Itis envisioned that any of the BoNT/B peptides disclosed in the presentspecification can be useful in a method of determining immunoresistanceto botulinum toxin therapy in an individual, with the proviso that theBoNT/B peptide can selectively bind to a anti-BoNT antibody.Non-limiting examples include a BoNT/B peptide derived from a naturallyoccurring BoNT/B, such as, e.g., the BoNT/B of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO:13, a BoNT/B isoform or a BoNT/B subtype; and a BoNT/B peptide derivedfrom a non-naturally occurring BoNT/B, such as, e.g., a conservativeBoNT/B variant, a non-conservative BoNT/B variant and a chimeric BoNT/Bpeptide. BoNT/B peptides disclosed in the present specification can beselected, e.g., depending on immunological factors, such as theselectivity of the BoNT/B peptide for an anti-BoNT antibody, andtechnical factors, such as chemical synthesis yields. It is alsounderstood that the two or more different BoNT/B peptides can beprovided separately or as part of a compound molecule such as a chimericBoNT/B peptide.

Aspects of the present invention provide, in part, a sample. As usedherein, the term “sample” means any biological matter that contains orpotentially contains at least one anti-BoNT antibody. An anti-BoNTantibody can be a neutralizing anti-BoNT antibody or a non-neutralizinganti-BoNT antibody. As used herein, the term “neutralizing anti-BoNTantibodies” means any anti-BoNT antibody that will, under physiologicalconditions, bind to a region of a BoNT toxin in such a manner as toreduce or prevent the toxin from exerting its effect in a BoNT therapy.Non-limiting examples of a neutralizing anti-BoNT antibody include,e.g., a neutralizing anti-BoNT/A antibody, a neutralizing anti-BoNT/Bantibody, a neutralizing anti-BoNT/C1 antibody, a neutralizinganti-BoNT/D antibody, a neutralizing anti-BoNT/E antibody, aneutralizing anti-BoNT/F antibody and a neutralizing anti-BoNT/Gantibody. As used herein, the term “non-neutralizing anti-BoNTantibodies” means any anti-BoNT antibody that will, under physiologicalconditions, bind to a region of a BoNT toxin, but not prevent the toxinfrom exerting its effect in a BoNT therapy. Non-limiting examples of anon-neutralizing anti-BoNT antibody include, e.g., a non-neutralizinganti-BoNT/A antibody, a non-neutralizing anti-BoNT/B antibody, anon-neutralizing anti-BoNT/C1 antibody, a non-neutralizing anti-BoNT/Dantibody, a non-neutralizing anti-BoNT/E antibody, a non-neutralizinganti-BoNT/F antibody and a non-neutralizing anti-BoNT/G antibody. It isenvisioned that any and all samples that can contain anti-BoNTantibodies can be used in this method, including, without limitation,blood, plasma, serum and lymph fluid. As used herein, the term “blood”means a bodily fluid including a cellular component and plasma andencompasses both whole blood and a blood component thereof. In addition,any and all individuals capable of raising anti-BoNT antibodies againsta BoNT toxin can serve as a source for a sample including, but notlimited to, birds and mammals, including mice, rats, goats, sheep,horses, donkeys, cows, primates and humans. Non-limiting examples ofspecific protocols for blood collection and serum preparation aredescribed in, e.g., Marjorie Schaub Di Lorenzo & Susan King Strasinger,BLOOD COLLECTION IN HEALTHCARE (F. A. Davis Company, 2001); and DianaGarza & Kathleen Becan-McBride, PHLEBOTOMY HANDBOOK: BLOOD COLLECTIONESSENTIALS (Prentice Hall, 6^(th) ed., 2002). These protocols areroutine procedures well within the scope of one skilled in the art andfrom the teaching herein.

A sample can be a test sample, or a sample can be a control sample. Asused herein, the term “test sample” means any sample in which thepresence or absence of an anti-BoNT antibody is sought to be determined.A test sample can be obtained from an individual prior to exposure to aBoNT toxin, after a single BoNT treatment, after multiple BoNT toxintreatments, before onset of resistance to a BoNT therapy, or after onsetof resistance to a BoNT therapy. As used herein, the term “controlsample” means any sample in which the presence or absence of ananti-BoNT antibody is known, and includes both negative and positivecontrol samples. A negative control sample can be obtained from anindividual who was never exposed to BoNT toxin and may include, withoutlimitation, a sample from the same individual supplying the test sample,but taken before undergoing a BoNT therapy; a sample taken from adifferent individual; a pooled sample taken from a plurality ofdifferent individuals. A positive control sample can be obtained from anindividual manifesting BoNT immunoresistance and includes, withoutlimitation, samples testing positive in a patient-based testing assays;samples testing positive in an in vivo bioassay; and samples showinghyperimmunity against an anti-BoNT antiserum.

Any of the above methods of the invention can be practiced, if desired,by selectively determining the presence or absence in the individual ofIgG antibodies immunoreactive with each BoNT/B peptide. Thus, it isforeseen that anti-BoNT antibodies can be purified from a sample.Anti-BoNT antibodies can be purified from a sample, using a variety ofprocedures including, without limitation, Protein A/G chromatography andaffinity chromatography. Non-limiting examples of specific protocols forpurifying antibodies from a sample are described in, e.g., ANTIBODIES: ALABORATORY MANUAL (Edward Harlow & David Lane, eds., Cold Spring HarborLaboratory Press, 2 ed. 1998); USING ANTIBODIES: A LABORATORY MANUAL:PORTABLE PROTOCOL No. I (Edward Harlow & David Lane, Cold Spring HarborLaboratory Press, 1998); and MOLECULAR CLONING, A LABORATORY MANUAL,supra, (2001). In addition, non-limiting examples of antibodypurification methods as well as well-characterized reagents, conditionsand protocols are readily available from commercial vendors thatinclude, without limitation, Pierce Biotechnology, Inc., Rockford, Ill.;and Zymed Laboratories, Inc., South San Francisco, Calif. Theseprotocols are routine procedures well within the scope of one skilled inthe art.

Techniques for determining a level of IgG antibodies immunoreactive witha BoNT/B peptide are well known in the art. For example, a solid-phaseradioimmunoassay for IgG anti-BoNT antibodies can be performed using ananti-mouse IgG secondary antibody. A variety of additional anti-IgGantibodies, including anti-human IgG antibodies, are well known in theart and are commercially available, including, but not limited to,rabbit anti-human IgG from Bethyl Laboratories, Inc. (Montgomery, Tex.)and goat anti-human IgG from Zymed Laboratories, Inc (San Francisco,Calif.). Thus, the methods of the invention can be practiced using anyof the immunoassays described hereinabove or well known in the art whichare specific for detection of IgG antibodies, for example, through useof an anti-IgG secondary antibody.

The present invention additionally provides a method of determiningimmunoresistance to botulinum toxin therapy in an individual bydetermining the level of IgG antibodies immunoreactive with thebotulinum toxin in the individual; and comparing the level of IgGantibodies to a control level of IgG antibodies, where an increase inthe level of IgG antibodies in the individual as compared to the controllevel indicates immunoresistance to the botulinum toxin therapy. Such anincrease can be, for example, at least a 5-fold increase or at least a10-fold increase. In one embodiment, the control level of IgG antibodiesis determined in a individual that has not been treated with botulinumtoxin therapy. In another embodiment, the control level of IgGantibodies is determined in an individual that is responsive to thebotulinum toxin therapy. The methods of the invention can be used todetermine immunoresistance to any of several botulinum toxin therapiesincluding, without limitation, a BoNT/A therapy, a BoNT/B therapy, aBoNT/C1 therapy, a BoNT/D therapy, a BoNT/E therapy, a BoNT/F therapyand a BoNT/G therapy.

Thus, an embodiment, the sample comprises blood. In aspect of thisembodiment, the sample comprises mouse blood, rat blood, goat blood,sheep blood, horse blood, donkey blood, cow blood, primate blood andhuman blood. In another embodiment, the sample comprises plasma. Inaspect of this embodiment, the sample comprises mouse plasma, ratplasma, goat plasma, sheep plasma, horse plasma, donkey plasma, cowplasma, primate plasma and human plasma. In another embodiment, thesample comprises serum. In aspect of this embodiment, the samplecomprises mouse serum, rat serum, goat serum, sheep serum, horse serum,donkey serum, cow serum, primate serum and human serum. In anotherembodiment, the sample comprises lymph fluid. In aspect of thisembodiment, the sample comprises mouse lymph fluid, rat lymph fluid,goat lymph fluid, sheep lymph fluid, horse lymph fluid, donkey lymphfluid, cow lymph fluid, primate lymph fluid and human lymph fluid. Inyet another embodiment, the sample is a test sample. In yet anotherembodiment, the sample is a control sample.

Aspects of the present invention provide, in part, determining thepresence or absence of anti-BoNT antibodies immunoreactive with a BoNT/Bpeptide. In is envisioned that any and all assay formats suitable forindicating the presence or absence of anti-BoNT antibody-BoNT/B peptidecomplexes and, therefore, to determine immunoresistance to botulinumtoxin therapy according to a method of the present invention. Such assayformats generally involve detecting an antigen-antibody interaction.Non-limiting examples include radioimmunoassays, enzyme-linkedimmunosorbent assays, enzyme immunoassays, fluorescence immunoassays,luminescent immunoassays and other nonradioisotopic assay formats, seee.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra, 2001; and CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, supra, 2004. Non-competitive assays canbe performed, for example, by attaching one or more selected BoNT/Bpeptides to a solid support; adding a test specimen; adding a secondaryantibody, which is an antibody selective for the test antibody; anddetecting the secondary antibody, typically by a physical property orenzymatic activity of the secondary antibody. In such an assay, theamount of signal that is detected can be proportional to the amount ofantibodies which are immunoreactive with the one or more BoNT/B peptidesand are present in the test specimen.

It is further foreseen that an assay format can either qualitatively orquantitatively determine the presence of an anti-BoNT antibody-BoNT/Bpeptide complex. Qualitative measurements can be determined by a widevariety of methods, such as, e.g., audioradiography, immunoblottingtechniques, and the like. Quantitative measurements can be determined bya wide variety of methods, such as, e.g., scintillation counters,spectrophotometers, densitometers, fluorometers, spectroluminometers,luminometers, high pressure liquid chromatography, and the like. Inaddition, control samples can also be assayed with a test sample usingthis method in order to provide baseline values useful for comparisonswith a test sample. Thus, a negative control comprises a sample knownnot to contain any anti-BoNT antibodies. A negative control canestablish a parameter for background noise levels and provide a means todistinguish false positive results from an actual BoNT immune resistanceresponse. A sample known to contain high levels of neutralizinganti-BoNT antibodies from an individual diagnosed with BoNTimmunoresistance could serve as a positive control. A positive controlcan provide a parameter from which a test sample can be evaluated todetermine the relative severity of immunoresistance occurring in a testpatient. One skilled in the art understands that, if desired, aquantitative method can be used for qualitative measurements. Inaddition, one skilled in the art understands that the selection of amethod of measurement is determined by the detection means employed.

In one aspect of the present invention, all steps of a method fordetermining the presence or absence of an anti-BoNT antibody areperformed in solution. In other aspects of the method disclosed in thepresent specification, it is also envisioned that a method canoptionally attach an assay component to a solid or insoluble material.Such a solid support can be, without limitation, e.g., a tube; plate;pins or “dipsticks”, column; particle, bead or other spherical orfibrous chromatographic media, such as, e.g., agarose beads, sepharosebeads, silica beads and plastic beads; sheets or membranes, such as,e.g., nitrocellulose and polyvinylidene fluoride (PVDF). The solidsupport selected can have a physical property that renders it readilyseparable from soluble or unbound material and generally allows unboundmaterials, such as, e.g., unbound antibodies, excess reagents, reactionby-products, or solvents, to be separated or otherwise removed (by,e.g., washing, filtration, centrifugation, etc.) from solidsupport-bound assay components. Non-limiting examples of how to make anduse a solid support-bound assay component are described in, e.g.,MOLECULAR CLONING, A LABORATORY MANUAL, supra, 2001; and CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, supra, 2004.

As a general procedure, because the exact amount of a BoNT/B peptide canbe readily determined by one skilled in the art, the assay amounts of asample and an anti-BoNT antibody can be determined based on a fixedassay amount of a BoNT/B peptide.

In an embodiment, it is envisioned that detecting the presence of anyand all binding levels of a BoNT/B peptide to an anti-BoNT antibodycapable of being detected by an assay format disclosed in the presentspecification are useful in aspects of the present invention. Thus,aspects of this embodiment may include detecting the presence of, e.g.,at least 10% complex formation of a BoNT/B peptide with an anti-BoNTantibody, at least 20% complex formation of a BoNT/B peptide with ananti-BoNT antibody, at least 30% complex formation of a BoNT/B peptidewith an anti-BoNT antibody, at least 40% complex formation of a BoNT/Bpeptide with an anti-BoNT antibody, at least 50% complex formation of aBoNT/B peptide with an anti-BoNT antibody, at least 60% complexformation of a BoNT/B peptide with an anti-BoNT antibody, at least 70%complex formation of a BoNT/B peptide with an anti-BoNT antibody, atleast 80% complex formation of a BoNT/B peptide with an anti-BoNTantibody, or at least 90% complex formation of a BoNT/B peptide with ananti-BoNT antibody. In other aspects of this embodiment may includedetecting the presence of, e.g., at most 10% complex formation of aBoNT/B peptide with an anti-BoNT antibody, at most 20% complex formationof a BoNT/B peptide with an anti-BoNT antibody, at most 30% complexformation of a BoNT/B peptide with an anti-BoNT antibody, at most 40%complex formation of a BoNT/B peptide with an anti-BoNT antibody, atmost 50% complex formation of a BoNT/B peptide with an anti-BoNTantibody, at most 60% complex formation of a BoNT/B peptide with ananti-BoNT antibody, at most 70% complex formation of a BoNT/B peptidewith an anti-BoNT antibody, at most 80% complex formation of a BoNT/Bpeptide with an anti-BoNT antibody, or at most 90% complex formation ofa BoNT/B peptide with an anti-BoNT antibody. To ascertain an appropriateassay amount of a BoNT/B peptide in this embodiment, the bindingcapacity of a BoNT/B peptide preparation towards an anti-BoNT antibodyis determined using a fixed amount of an anti-BoNT antibody and a rangeof BoNT/B peptide amounts in order to generate an saturation curve foranti-BoNT antibody-BoNT/B peptide complex formation. This protocol isroutine procedure well within the scope of one skilled in the art andfrom the teaching herein.

In yet another embodiment, a wide range of BoNT/B peptide amounts can beused in methods disclosed in the present specification. The assay amountof a BoNT/B peptide can be varied as appropriate by one skilled in theart and generally depends, in part, on the amount of anti-BoNTantibodies used, the volume of sample used and the assay formatemployed. Therefore, aspects of this embodiment may include a BoNT/Bpeptide amount of, e.g., at least 1 ng, at least 10 ng, at least 100 ng,at least 1 μg, at least 2.5 μg, at least 5.0 μg or at least 10 μg. Inother aspects of this embodiment may include a BoNT/B peptide amount of,e.g., at most 1 ng, at most 10 ng, at most 100 ng, at most 1 μg, at most2.5 μg, at most 5.0 μg or at most 10 μg. In an aspect of thisembodiment, the assay amount of a BoNT/B peptide is 100 ng. In anotheraspect of this embodiment, the assay amount of a BoNT/B peptide is 1 μg.In another aspect of this embodiment, the assay amount of a BoNT/BApeptide is 2.5 μg.

In yet another embodiment of the present invention, a wide range ofsample volumes can be used in methods disclosed in the presentspecification. The assay amount of a sample can be varied as appropriateby one skilled in the art and generally depends, in part, on the amountof sample available, the BoNT/B peptide amount being used, the anti-BoNTantibody amount present in a sample and the assay format employed. Thus,aspects of this embodiment a sample volume can include, e.g., at least 1μL, at least 2.5 μL, at least 5 μL, at least 10 μL, at least 20 μL, atleast 30 μL, at least 40 μL, at least 50 μL, or at least 100 μL. Inother aspects of this embodiment a sample volume can include, e.g., atmost 1 μL, at most 2.5 μL, at most 5 μL, at most 10 μL, at most 20 μL,at most 30 μL, at most 40 μL, at most 50 μL, or at most 100 μL. In anaspect of this embodiment, the assay amount of a sample is 50 μL.

In still another embodiment, it is envisioned that a wide range of assayvolumes can be used in methods disclosed in the present specification.Thus aspects of this embodiment an assay volume can include, e.g. atleast, 1 μL, at least 2 μL, at least 3 μL, at least 4 μL, at least 5 μL,at least 10 μL, at least 20 μL, at least 30 μL, at least 40 μL, at least50 μL, at least 100 μL, at least 200 μL, at least 300 μL, at least 400μL, at least 500 μL, or at least 1000 μL. In other aspects of thisembodiment assay volume can include, e.g. at most 1 μL, at most 2 μL, atmost 3 μL, at most 4 μL, at most 5 μL, at most 10 μL, at most 20 μL, atmost 30 μL, at most 40 μL, at most 50 μL, at most 100 μL, at most 200μL, at most 300 μL, at most 400 μL, at most 500 μL, or at most 1000 μL.

In still another embodiment, it is envisioned that any and alltemperatures that allow the formation of an anti-BoNT antibody-BoNT/Bpeptide complex can be used in methods disclosed in the presentspecification. Assay temperatures can be varied as appropriate by oneskilled in the art and generally depend, in part, on the BoNT/B peptideamount, the anti-BoNT antibody amount present in a sample, the samplevolume used, the assay volume used, the assay format employed and assaytime. Thus, an assay temperature should not be as low as to cause thesolution to freeze and should not be as high as to denature the BoNT/Bpeptides, anti-BoNT antibodies or other proteins disclosed in thepresent specification. In an aspect of this embodiment, the assay isperformed within a temperature range above 0° C., but below 40° C. Inanother aspect of this embodiment, the assay is performed within atemperature range of about 4° C. to about 37° C. In yet another aspectof this embodiment, the assay is performed within a temperature range ofabout 2° C. to 10° C. In yet another aspect of this embodiment, theassay is performed at about 4° C. In still another aspect of thisembodiment, the assay is performed within a temperature range of about10° C. to about 18° C. In still another aspect of this embodiment, theassay is performed at about 16° C. In yet another aspect of thisembodiment, the assay is performed within a temperature range of about18° C. to about 32° C. In yet another aspect of this embodiment, theassay is performed at about 20° C. In another aspect of this embodiment,the assay is performed within a temperature range of about 32° C. toabout 40° C. In another aspect of this embodiment, the assay isperformed at about 37° C.

In still another embodiment, it is foreseen that any and all timessufficient for the formation of an anti-BoNT antibody-BoNT/B peptidecomplex can be used in methods disclosed in the present specification.Assay times can be varied as appropriate by one skilled in the art andgenerally depend, in part, on the BoNT/B peptide amount, the anti-BoNTantibody amount present in a sample, the sample volume used, the assayvolume used, the assay format employed and the incubation temperature.Therefore, aspects of this embodiment include assay times of, e.g., atleast 1 minute, at least 2 minutes, at least 3 minutes, at least 4minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes,at least 20 minutes, at least 25 minutes, at least 30 minutes, at least45 minutes, at least 60 minutes, or at least 120 minutes. It isunderstood by one skilled in the art that an assay temperature canaffect the formation of an anti-BoNT antibody-BoNT/B peptide complexdisclosed in the present invention, and thereby can influence the lengthof time required to achieve sufficient complex formation. Thus, in anaspect of this embodiment, assay times of at least 45 minutes are usedin an assay temperature range of about 2° C. to about 10° C. In anotheraspect of this embodiment, assay times of at least 30 minutes are usedin an assay temperature range of about 10° C. to about 18° C. In yetanother aspect of this embodiment, assay times of at least 15 minutesare used in an assay temperature range of about 18° C. to about 32° C.In another aspect of this embodiment, assay times of at least 5 minutesare used in an assay temperature range of about 32° C. to about 40° C.In another aspect of this embodiment, an assay time of 15 minutes isused at an assay temperature of about 37° C.

In a further embodiment, it is also envisioned that any and all buffersthat allow the formation of an anti-BoNT antibody-BoNT/B peptide complexcan optionally be used in methods disclosed in the presentspecification. Assay buffers can be varied as appropriate by one skilledin the art and generally depend, in part, on the pH value desired forthe assay format employed, the BoNT/B peptide, the anti-BoNT antibodyand the assay format employed. Therefore, aspects of this embodiment mayoptionally include, e.g., 2-amino-2-hydroxymethyl-1,3-propanediol (Tris)buffers; Phosphate buffers, such as, e.g., potassium phosphate buffersand sodium phosphate buffers; Good buffers, such as, e.g.,2-(N-morpholino) ethanesulfonic acid (MES),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N,N′-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino) propanesulfonic acid (MOPS), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO),N-tris(hydroxymethyl) methylglycine (Tricine),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS); saline buffers,such as, e.g., Phosphate-buffered saline (PBS), HEPES-buffered saline,and Tris-buffered saline (TBS); Acetate buffers, such as, e.g.,magnesium acetate, potassium actetate, and Tris acetate; and the like,or any combination thereof. In addition, the buffer concentration in amethod disclosed in the present specification can be varied asappropriate by one skilled in the art and generally depend, in part, onthe buffering capacity of a particular buffer being used and thedetection means employed. Thus, aspects of this embodiment may include abuffer concentration of, e.g., at least 1 mM, at least 5 mM, at least 10mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, atleast 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, or at least100 mM. Non-limiting examples of how to make and use specific buffersare described in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra,(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004).

In a further embodiment, it is also envisioned that any and all saltsthat allow the formation of an anti-BoNT antibody-BoNT/B peptide complexcan optionally be used in methods disclosed in the presentspecification. Assay salts can be varied as appropriate by one skilledin the art and generally depend, in part, on the physiologicalconditions desired for the assay format employed, the BoNT/B peptide,the anti-BoNT antibody and the assay format employed. Therefore, aspectsof this embodiment may optionally include, e.g., sodium chloride,potassium chloride, calcium chloride, magnesium chloride, manganesechloride, zinc chloride, magnesium sulfate, zinc sulfate, and the like,or any combination thereof. In addition, the salt concentration in amethod disclosed in the present specification can be varied asappropriate by one skilled in the art and generally depend, in part, onthe buffering capacity of a particular buffer being used and thedetection means employed. Thus, aspects of this embodiment may include asalt concentration of, e.g., at least 1 mM, at least 5 mM, at least 10mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, atleast 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, or at least100 mM. Non-limiting examples of how to make and use specific salts aredescribed in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra,(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004).

In a further embodiment, it is also envisioned that any and allenhancing agents that allow the formation of an anti-BoNTantibody-BoNT/B peptide complex can optionally be used in methodsdisclosed in the present specification. Assay enhancing agents can bevaried as appropriate by one skilled in the art and generally depend, inpart, on the assay conditions desired for the assay format employed, theBoNT/B peptide, the anti-BoNT antibody and the assay format employed.Therefore, aspects of this embodiment may optionally include, e.g.,stabilizing agents including proteins, such as, e.g., bovine serumalbumin and milk proteins, such as, e.g., casein, thyroglobulin, fetuin,asialofetuin, cytochrome c and bovine submaxillary mucin and polyamines,such as, e.g., spermidine and spermine; chelating agents including,e.g., ethylenediamine tetraacetic acid (EDTA) and ethylene glycolbis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA); reducingagents, including, e.g., -mercaptoethanol and dithiothreitol (DTT);dimethylsulfoxide (DMSO); and the like, or any combination thereof. Inaddition, the enhancing agent concentration in a method disclosed in thepresent specification can be varied as appropriate by one skilled in theart and generally depend, in part, on the assay conditions desired forthe assay and the detection means employed. In an aspect of thisembodiment, concentrations for a stabilizing agent may include, e.g., atleast 10 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 200μg/mL or at least 500 μg/mL. In another aspect of this embodiment,concentrations for a chelating agent may include, e.g., at least 10 nM,at least 50 nM, at least 100 nM, at least 500 nM, at least 1 mM or atleast 10 mM. In yet another aspect of this embodiment, concentrationsfor a reducing agent may include, e.g., at least 10 nM, at least 50 nM,at least 100 nM, at least 500 nM, at least 1 mM, at least 10 mM or atleast 100 mM. Non-limiting examples of how to make and use specificenhancing agents are described in, e.g., MOLECULAR CLONING, A LABORATORYMANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,supra, (2004).

In an additional embodiment of the invention, it is also foreseen that awide variety of processing formats can be used in conjunction with themethods of the present invention, including, without limitation, manualprocessing, partial automated-processing, semi-automated-processing,full automated-processing, high throughput processing, high contentprocessing, and the like or any combination thereof.

It is understood by one skilled in the art that a wide variety offactors can influence assay conditions, including, without limitation,solution variations, buffer variations, reagent variations, equipmentvariations and facility variations. Thus, any particular assay conditionselected by one skilled in the art will require routine experimentationin order to optimize the method to account for such factors. Theseoptimization protocols are routine procedures well within the scope ofone skilled in the art and the teaching herein.

As a non-limiting example, a competitive assay can be performed byattaching one or more selected BoNT/B peptides to a solid support;adding simultaneously a test specimen and an enzyme-labeled secondaryantibody; and adding a substrate that produces a detectable compoundwhen acted upon by the enzyme. In this type of assay format, the amountof signal that is detected is inversely proportional to the amount ofanti-BoNT antibody present in the test specimen.

In one embodiment, the presence or absence of anti-BoNT antibodiesimmunoreactive with a BoNT/B peptide is determined using anenzyme-linked immunosorbent assay (ELISA). In another embodiment, thepresence or absence of anti-BoNT antibodies immunoreactive with a BoNT/Bpeptide is determined using a radioimmunoassay.

Various detection methods can be employed in any of the assay formatsdisclosed in the present specification, including without limitation, aradiation detection method, a fluorescence detection method, afluorescence resonance energy transfer (FRET) detection method, aphosphorescence detection method, a chemiluminescence detection method,a bioluminescence detection method, an electrochemiluminescencedetection method, a chromagenic detection method and an enzyme-activitydetection method. In addition, any of a variety of marker compoundssuitable for the detection system selected, can be operably-linked to aBoNT/B peptide as a labeled molecule including, without limitation, aradioisotope, fluorescent compound, a phosphorescent compound, achemiluminescent compound, a bioluminescent compound, and the like.Thus, in one aspect of the present invention, a marker compound suitablefor the selected detection system is operably-linked to a BoNT/B peptideas the labeled molecule suitable for any method. As used herein, theterm “operably linked” when used in reference to a labeled molecule,means any of a variety of chemical reactions that can join a markercompound disclosed in the present specification to a BoNT/B peptidedisclosed in the present specification such that a single peptide,comprising a peptide and marker compound, suitable to perform a methoddisclosed in the present specification is produced.

Non-limiting examples of radioisotopes that may be operably-linked to aBoNT/B peptide disclosed in the specification include, e.g., ³Hydrogen,Carbon, Sodium, Phosphorus, Phosphorus, ³⁵Sulfer, ³⁶-Chlorine,⁴⁵Calcium, 51 Chromium, ⁵⁷Cobalt, ⁵⁸Cobalt, ⁵⁹Iron, ⁶³Nickel, ⁶⁵Zinc,⁷⁵Selenium, ⁸⁶Rubidum, ¹⁰³Ruthenium, ¹⁰⁹Cadmium, ¹²⁵Iodine, ¹³Iodine,and the like. Non-limiting examples of fluorescent compounds that may beoperably-linked to a BoNT/B peptide disclosed in the specificationinclude, e.g., fluorescein, fluorescamine, isocyanate, isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde,Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532,Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594,Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680,Alexa Fluor® 700, Alexa Fluor® 750, Cy-2, Cy-3, Cy-5, Cy-7 and the like.Non-limiting examples of chemiluminescent compounds that may beoperably-linked to a BoNT/B peptide disclosed in the specificationinclude, e.g., imidazoles, such as, e.g., lophine; acylhydrazines, suchas, e.g., luminal and isoluminol; acridinium salts and esters, such as,e.g., lucigenin; oxalate salts and esters, such as, e.g.,bis(2,4,6-trichloropheryl) oxalate (TCPO) and bis(2,4,-dinitrophenyl)oxalate (DNPO); Tris (2,2N-bipyridine) ruthenium compounds, such as,e.g., ruthenium(bipyridine)₃, and the like. Non-limiting examples ofbioluminescent compounds that may be operably-linked to a BoNT/B peptidedisclosed in the specification include, e.g., bacterial luciferins,dinoflagellate luciferins, vargulins, porichthys luciferins,coelenterazines, beetle luciferins, 4-methylumbelliferone esters, andthe like.

Likewise, any of a variety of peptides suitable for the detection methodselected, can be operably-linked to a BoNT/B peptide as a fusion proteinincluding, without limitation, a peptide necessary for producingflorescence, a peptide necessary for producing phosphorescence, apeptide necessary for producing chemiluminescence, a peptide necessaryfor producing bioluminescence, and the like. As used herein, the term“operably linked” when used in reference to a fusion protein, means anyof a variety of cloning methods that can join a first nucleic acidsequence composition encoding a first peptide disclosed in the presentspecification in-frame with a second nucleic acid sequence compositionencoding a second peptide disclosed in the present specification suchthat a single peptide, comprising both the first and second peptides,suitable to perform a method disclosed in the present specification isproduced when expressed. In one embodiment, a peptide suitable for thedetection method selected, is operably-linked to a BoNT/B peptide.

Non-limiting examples of a peptide necessary for producing florescencethat may be operably-linked to a BoNT/B peptide disclosed in thespecification include, e.g., photoproteins, such as, e.g., aequorin;obelin; Aequorea fluorescent proteins, such, e.g., green fluorescentprotein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein(BFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),ultraviolet fluorescent protein (GFPuv), their fluorescence-enhancementvariants, including EGFP, ECFP, EBFP and EYFP, their peptidedestabilization variants, and the like; and Red coral reef fluorescentproteins (RCFPs), such, e.g., Discosoma red fluorescent protein (DsRed),Anemonia red fluorescent protein (AsRed), Heteractis far-red fluorescentprotein (HcRed), Anemonia cyan fluorescent protein (AmCyan), Zoanthusgreen fluorescent protein (ZsGreen), Zoanthus yellow fluorescent protein(ZsYellow), their fluorescence-enhancement variants, including DsRed2,AsRed2, their peptide destabilization variants, and the like.Non-limiting examples of a peptide necessary for producingchemiluminescence that may be operably-linked to a BoNT/B peptidedisclosed in the specification include, e.g., alkaline phosphatases,horseradish peroxidases, xanthine oxidases, glucose oxidases andβ-galatosidases. Non-limiting examples of a peptide necessary forproducing bioluminescence that may be operably-linked to a BoNT/Bpeptide disclosed in the specification include, e.g., bacterialluciferases, dinoflagellate luciferases, vargula luciferases,coelenterate luciferases, beetle luciferases, and the like. Non-limitingexamples of a peptide necessary for producing chromogenic compound thatmay be operably-linked to a BoNT/B peptide disclosed in thespecification include, e.g., alkaline phosphatases, horseradishperoxidases, ureases, β-glucourinidases, glucose oxidases andβ-galatosidases.

Non-limiting examples of specific protocols for selecting, making andusing detection systems, making and using peptides labeled with a markercompound and making and using fusion proteins are described in, e.g.,MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); METHODS INENZYMOLOGY, VOL. 305, BIOLUMINESCENCE AND CHEMILUMINESCENCE, PART C(Miriam M. Ziegler & Thomas O. Baldwin eds., Academic Press, 2000); Y.Fuster Mestre et al., Flow-chemiluminescence: A Growing Modality ofPharmaceutocal Analysis, 16 LUMINESCENCE 213-235, (2001); Lee F. GreerIII & Aladar A. Szalay, Imaging of Light Emission From the Expression ofLuciferases in Living Cells and Organisms: A Review, 17 LUMINESCENCE43-74, (2002); Richard W. Horobin & John A. Kiernan, CONN'S BIOLOGICALSTAINS: A HANDBOOK OF DYES, STAINS AND FLUOROCHROMES FOR USE IN BIOLOGYAND MEDICINE (BIOS Scientific Publishers, 10^(th) ed. 2002); HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS, http://www.probes.com/handbook (Molecular Probes, Inc., 9^(th) ed, 2004), and CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). In addition, non-limitingexamples of how to make and use detection systems, labeled peptides andfusion protein disclosed in the present specification, as well aswell-characterized reagents, conditions and protocols are readilyavailable from commercial vendors that include, without limitation,Amersham Biosciences, Piscataway, N.J.; BD Biosciences-Clontech, PaloAlto, Calif.; Bio-Rad Laboratories, Hercules, Calif.; Cayman ChemicalCo., Ann Arbor, Mich.; Molecular Probes, Inc., Eugene, Oreg.;PerkinElmer Life and Analytical Sciences, Inc., Boston, Mass.; PierceBiotechnology, Inc., Rockford, Ill.; Princeton Separations, Adelphia,N.J.; and Vector Laboratories, Burlingame, Calif. These protocols areroutine procedures within the scope of one skilled in the art and fromthe teaching herein.

Thus, in an embodiment, a BoNT/B peptide is operably-linked to aradioisotope. In an aspect of this embodiment, a BoNT/B peptide isoperably-linked to ³Hydrogen, ¹⁴-Carbon, ²²Sodium, ³²Phosphorus,³³Phosphorus, ³⁵Sulfer, ³⁶-Chlorine, ⁴⁵Calcium, ⁵¹Chromium, ⁵⁷Cobalt,⁵⁸Cobalt, ⁵⁹Iron, ⁶³Nickel, ⁶⁵Zinc, ⁷⁵Selenium, ⁸⁶Rubidum, ¹⁰³Ruthenium,¹⁰⁹Cadmium, ¹²⁵Iodine or ¹³¹Iodine. In another aspect of thisembodiment, the presence of anti-BoNT antibody-BoNT/B peptide complexesis quantitatively determined using a scintillation counter. In anotheraspect of this embodiment, the absence of anti-BoNT antibody-BoNT/Bpeptide complexes is quantitatively determined using a scintillationcounter.

In yet another embodiment, a BoNT/B peptide is operably-linked to afluorescent compound. In an aspect of this embodiment, a BoNT/B peptideis operably-linked to a fluorescein, a fluorescamine, an isocyanate, anisothiocyanate, a rhodamine, a phycoerythrin, a phycocyanin, anallophycocyanin, an o-phthaldehyde, an Alexa Fluor® 350, an Alexa Fluor®430, an Alexa Fluor® 488, an Alexa Fluor® 532, an Alexa Fluor® 546, anAlexa Fluor® 555, an Alexa Fluor® 568, an Alexa Fluor® 594, an AlexaFluor® 633, an Alexa Fluor® 647, an Alexa Fluor® 660, an Alexa Fluor®680, an Alexa Fluor® 700, an Alexa Fluor® 750, a Cy-2, a Cy-3, a Cy-5 ora Cy-7. In another aspect of this embodiment, the presence of anti-BoNTantibody-BoNT/B peptide complexes is quantitatively determined using aspectrofluorometer (see, e.g., Example 7). In another aspect of thisembodiment, the absence of anti-BoNT antibody-BoNT/B peptide complexesis quantitatively determined using a spectrofluorometer.

In another embodiment, a BoNT/B peptide is operably-linked to aphotoprotein. In an aspect of this embodiment, a BoNT/B peptide isoperably linked to an aequorin, an obelin, a GFP, an EGFP, a CFP, anECFP, a BFP, an EBFP, a YFP, an EYFP, a GFPuv, a DsRed, a DsRed2, aAsRed, a AsRed2, a HcRed, an AmCyan, a ZsGreen or a ZsYellow. In anotheraspect of this embodiment, the presence of anti-BoNT antibody-BoNT/Bpeptide complexes is quantitatively determined using aspectrofluorometer.

In another aspect of this embodiment, the absence of anti-BoNTantibody-BoNT/B peptide complexes is quantitatively determined using aspectrofluorometer.

In yet another embodiment, a BoNT/B peptide is operably-linked to achemiluminescent compound. In an aspect of this embodiment, a BoNT/Bpeptide is operably linked to an imidazole, an acridinium salt, anacridinium ester, an oxalate salt, an oxalate ester, or a Tris(2,2N-bipyridine) ruthenium compound. In another aspect of thisembodiment, the presence of anti-BoNT antibody-BoNT/B peptide complexesis quantitatively determined using a luminometer. In another aspect ofthis embodiment, the absence of anti-BoNT antibody-BoNT/B peptidecomplexes is quantitatively determined using a luminometer.

In yet another embodiment, a BoNT/B peptide is operably-linked to apeptide necessary for producing chemiluminescence. In an aspect of thisembodiment, a BoNT/B peptide is operably linked to an alkalinephosphatase, a horseradish peroxidase, a xanthine oxidase, a glucoseoxidase or a β-galactosidase. In another aspect of this embodiment, thepresence of anti-BoNT antibody-BoNT/B peptide complexes isquantitatively determined using a luminometer. In another aspect of thisembodiment, the absence of anti-BoNT antibody-BoNT/B peptide complexesis quantitatively determined using a luminometer.

In yet another embodiment, a BoNT/B peptide is operably-linked to apeptide necessary for producing bioluminescence. In an aspect of thisembodiment, a BoNT/B peptide is operably linked to a bacterialluciferase, a dinoflagellate luciferase, a vargula luciferase, acoelenterate luciferase or a beetle luciferase. In another aspect ofthis embodiment, the presence of anti-BoNT antibody-BoNT/B peptidecomplexes is quantitatively determined using a luminometer. In anotheraspect of this embodiment, the absence of anti-BoNT antibody-BoNT/Bpeptide complexes is quantitatively determined using a luminometer.

In yet another embodiment, a BoNT/B peptide is operably-linked to apeptide necessary for producing a chromogenic product. In an aspect ofthis embodiment, a BoNT/B peptide is operably linked to an alkalinephosphatase, a horseradish peroxidase, an urease, α-glucourinidase, aglucose oxidase or a β-galactosidase. In another aspect of thisembodiment, the presence of anti-BoNT antibody-BoNT/B peptide complexesis quantitatively determined using a spectrophotometer. In anotheraspect of this embodiment, the absence of anti-BoNT antibody-BoNT/Bpeptide complexes is quantitatively determined using aspectrophotometer.

Aspects of the present invention provide, in part, comparing the amountof anti-BoNT antibody-BoNT/B peptide complexes formed in the test sampleto the amount of anti-BoNT antibody-BoNT/B peptide complexes formed inthe control sample. In an embodiment, the amount of anti-BoNTantibody-BoNT/B peptide complexes in the test sample is increased ascompared to the amount of anti-BoNT antibody-BoNT/B peptide complexesformed in the control sample. In an aspect of this embodiment, anincrease in the amount of anti-BoNT antibody-BoNT/B peptide complexesformed in the test sample as compared to a positive control sampleindicates an increase of immunoresistance to a BoNT therapy in theindividual. In another aspect of this embodiment, an increase in theamount of anti-BoNT antibody-BoNT/B peptide complexes formed in the testsample as compared to a negative control sample indicates an increase ofimmunoresistance to a BoNT therapy in the individual. In anotherembodiment, the amount of anti-BoNT antibody-BoNT/B peptide complexesformed in the test sample is decreased as compared to the amount ofanti-BoNT antibody-BoNT/B peptide complexes formed in the controlsample. In an aspect of this embodiment, a decrease in the amount ofanti-BoNT antibody-BoNT/B peptide complexes formed in the test sample ascompared to a positive control sample indicates a decrease ofimmunoresistance to a BoNT therapy in the individual. In another aspectof this embodiment, a decrease in the amount of anti-BoNTantibody-BoNT/B peptide complexes formed in the test sample as comparedto a negative control sample indicates a decrease of immunoresistance toa BoNT therapy in the individual.

In an embodiment, the presence of anti-BoNT antibody-BoNT/B peptidecomplexes in the test sample indicates the presence of immunoresistanceto a BoNT therapy in the individual. In an aspect of this embodiment,the presence of anti-BoNT antibody-BoNT/B peptide complexes in the testsample is compared to the presence of anti-BoNT antibody-BoNT/B peptidecomplexes in the control sample. In an aspect of this embodiment, thepresence of anti-BoNT antibody-BoNT/B peptide complexes in the testsample as compared to a negative control sample indicates the presenceof immunoresistance to a BoNT therapy in the individual.

In another embodiment, the absence of anti-BoNT antibody-BoNT/B peptidecomplexes in the test sample indicates the absence of immunoresistanceto a BoNT therapy in the individual. In an aspect of this embodiment,the absence of anti-BoNT antibody-BoNT/B peptide complexes in the testsample is compared to the absence of anti-BoNT antibody-BoNT/B peptidecomplexes in the control sample. In another aspect of this embodiment,the absence of anti-BoNT antibody-BoNT/B peptide complexes in the testsample as compared to a positive control sample indicates the absence ofimmunoresistance to a BoNT therapy in the individual.

Aspects of the present invention provide, in part, comparing the amountof free or unbound BoNT/B peptides in the test sample to the amount offree or unbound BoNT/B peptides in the control sample. In an embodiment,the amount of free or unbound BoNT/B peptides in the test sampleincreases as compared to the amount of free or unbound BoNT/B peptidesin the control sample. In an aspect of this embodiment, an increase inthe amount of free or unbound BoNT/B peptides in the test sample ascompared to a positive control sample indicates a decrease ofimmunoresistance to a BoNT therapy in the individual. In another aspectof this embodiment, an increase in the amount of free or unbound BoNT/Bpeptides in the test sample as compared to a negative control sampleindicates a decrease of immunoresistance to a BoNT therapy in theindividual. In another embodiment, the amount of free or unbound BoNT/Bpeptides in the test sample decreases as compared to the amount of freeor unbound BoNT/B peptides in the control sample. In an aspect of thisembodiment, a decrease in the amount of free or unbound BoNT/B peptidesin the test sample as compared to a positive control sample indicates anincrease of immunoresistance to a BoNT therapy in the individual. Inanother aspect of this embodiment, a decrease in the amount of free orunbound BoNT/B peptides in the test sample as compared to a negativecontrol sample indicates an increase of immunoresistance to a BoNTtherapy in the individual.

Aspects of the present invention provide, in part, comparing the amountof free or unbound anti-BoNT antibodies in the test sample to the amountof free or unbound anti-BoNT antibodies in the control sample. In anembodiment, the amount of free or unbound anti-BoNT antibodies in thetest sample increases as compared to the amount of free or unboundanti-BoNT antibodies in the control sample. In an aspect of thisembodiment, an increase in the amount of free or unbound anti-BoNTantibodies in the test sample as compared to a positive control sampleindicates an increase of immunoresistance to a BoNT therapy in theindividual. In another aspect of this embodiment, an increase in theamount of free or unbound anti-BoNT antibodies in the test sample ascompared to a negative control sample indicates an increase ofimmunoresistance to a BoNT therapy in the individual. In anotherembodiment, the amount of free or unbound anti-BoNT antibodies in thetest sample decreases as compared to the amount of free or unboundanti-BoNT antibodies in the control sample. In an aspect of thisembodiment, a decrease in the amount of free or unbound anti-BoNTantibodies in the test sample as compared to a positive control sampleindicates a decrease of immunoresistance to a BoNT therapy in theindividual. In another aspect of this embodiment, a decrease in theamount of free or unbound anti-BoNT antibodies in the test sample ascompared to a negative control sample indicates a decrease ofimmunoresistance to a BoNT therapy in the individual.

Thus, in one embodiment, a method of determining immunoresistance tobotulinum toxin therapy in an individual comprising the step ofdetermining the presence or absence in the individual of anti-BoNTantibodies immunoreactive with a BoNT/B peptide, where the presence ofthe anti-BoNT antibody-BoNT/B peptide complex is indicative ofimmunoresistance to a BoNT therapy.

In another embodiment, a method of determining immunoresistance to BoNTtherapy in an individual, the method comprising the steps of combining aBoNT/B peptide and a test sample under conditions suitable for theselective binding of the BoNT/B peptide to an anti-BoNT antibody anddetermining the presence of an anti-BoNT antibody-BoNT/B peptidecomplex, the antibody-peptide complex formed by the selective binding ofan anti-BoNT antibody and the BoNT/B peptide, where the presence of theanti-BoNT antibody-BoNT/B peptide complex is indicative ofimmunoresistance to a BoNT therapy.

In another embodiment, a method of determining immunoresistance to BoNTtherapy in an individual, the method comprising the steps of combining aBoNT/B peptide and a test sample under conditions suitable for theselective binding of the BoNT/B peptide to an anti-BoNT antibody anddetermining the presence of an anti-BoNT antibody-BoNT/B peptidecomplex, the antibody-peptide complex formed by the selective binding ofan anti-BoNT antibody and the BoNT/B peptide and correlating the amountof an antibody-peptide complex formed from the test sample relative tothe amount of an antibody-peptide complex formed by the BoNT/B peptidecombined to a control sample where the presence of the anti-BoNTantibody-BoNT/B peptide complex is indicative of immunoresistance to aBoNT therapy.

Patients treated with a botulinum toxin therapy can developimmunoresistance to the therapeutic treatment, reducing or eliminatingthe beneficial effect of botulinum toxin therapy. Methods that preventor reduce the development of a BoNT-specific immune response in anindividual, which in turn can prevent or reduce immunoresistance to abotulinum toxin therapy, are of major importance. These treatments wouldallow for 1) the suppression of a potential deleterious immune responsein a patient undergoing BoNT therapy thereby affording a more prolongedtreatment course relative to current therapies; 2) the suppression of aBoNT immunoresponsive state in a patient thereby offering additionaltreatments that would otherwise have been ineffective. Therefore, theseassays present a major benefit in terms of providing better patient careand reducing health care costs. The BoNT/B peptides disclosed in thepresent specification are useful in methods of determiningimmunoresistance to botulinum toxin therapy in aindividual. Thesepeptides each contain one or more epitopes recognized by antibodiescontained in antisera from animals immunized with BoNT/B, and thus canserve as binding substrates for anti-BoNT/B antibodies. The methodsdisclosed in the present specification can be useful for preventing orreducing immunoresistance to any of a variety of botulinum toxintherapies including, but not limited to, a BoNT/A therapy, a BoNT/Btherapy, a BoNT/C1 therapy, a BoNT/D therapy, a BoNT/E therapy, a BoNT/Ftherapy and a BoNT/G therapy.

Thus, the present invention provides a method of treatingimmunoresistance to botulinum toxin therapy in an individual byadministering to the individual a tolerogizing composition comprising atolerogizing agent operably linked to a BoNT/B peptide theadministration preventing or reducing immunoresistance to botulinumtoxin therapy. Those skilled in the art can readily determine for aparticular tolerogizing composition, a suitable pharmacologicalcomposition, an appropriate antigen payload; route of administration;volume of dose; and tolerogizing regimen useful in a particularindividual, for example, humans.

Aspects of the present invention provide, in part, a method of treatingimmunoresistance to botulinum toxin therapy, such as, e.g., a BoNT/Aimmunoresistance condition, a BoNT/B immunoresistance condition, aBoNT/C1 immunoresistance condition, a BoNT/D immunoresistance condition,a BoNT/E immunoresistance condition, a BoNT/F immunoresistance conditionor a BoNT/G immunoresistance condition. As used herein, the term“treating,” when used in reference to administering to an individual atolerogizing composition, means reducing a symptom of a conditioncharacterized by resistance to a BoNT therapy, or delaying or preventingonset of a symptom of a condition characterized by a BoNTimmunoresistance in the individual. For example, the term “treating”means reducing a symptom of a condition characterized by a BoNTimmunoresistance by, e.g., at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 100%.The effectiveness of a tolerogizing composition in treating a conditioncharacterized by BoNT immunoresistance can be determined by observingone or more clinical symptoms or physiological indicators associatedwith the condition. An improvement in a condition characterized by BoNTimmunoresistance also can be indicated by a reduced need for aconcurrent therapy. Those of skill in the art will know the appropriatesymptoms or indicators associated with specific conditions and will knowhow to determine if an individual is a candidate for treatment with atolerogizing composition disclosed in the present specification. Inparticular, it is understood that those skilled in the art will be ableto determine if a condition is characterized by BoNT immunoresistance,e.g., by comparison of levels of BoNT immunoresistance from anindividual suspected to have an immunoresistance to a BoNT therapy withan individual not suspected to have an immunoresistance to a BoNTtherapy.

Aspects of the present invention provide, in part, administration of atolerogizing composition. As used herein, the term “administration”means any delivery mechanism that provides a tolerogizing composition toan individual that potentially results in a clinically, therapeutically,cosmetically or experimentally beneficial result. A tolerogizingcomposition useful in the methods of the invention can be administeredto an individual by any of a variety of routes depending, for example,on the type and location of BoNT immunoresistance to be treated, thetolerogizing composition, or other compound to be included in thecomposition, and the history, risk factors and symptoms of the subject.Routes of administration suitable for the methods of the inventioninclude both local and systemic administration. Local administrationresults in significantly more tolerogizing composition being deliveredto a specific location as compared to the entire body of the subject,whereas, systemic administration results in delivery of a tolerogizingcomposition to essentially the entire body of the subject. Atolerogizing composition can also be administered peripherally. As usedherein, the term “peripheral administration” or “administeredperipherally” means introducing an agent into an individual outside ofthe central nervous system. Peripheral administration encompasses anyroute of administration other than direct administration to the spine orbrain. As such, it is clear that intrathecal and epidural administrationas well as cranial injection or implantation is not within the scope ofthe term “peripheral administration” or “administered peripherally.”

Administration of a tolerogizing composition can be by a variety ofroutes including, without limitation, orally in any acceptable form,such as, e.g., tablet, liquid, capsule, powder, or the like; topicallyin any acceptable form, such as, e.g., patch, drops, creams, gels orointments; by injection, in any acceptable form, such as, e.g.,intravenous, intraperitoneal, intramuscular, subcutaneous, parenteral orepidural; and by implant, such as, e.g., subcutaneous pump, intrathecalpump, suppository, bioerodible delivery system, non-bioerodible deliverysystem or other implanted extended or slow release device orformulation. As a non-limiting example, oral tolerance iswell-recognized in the art (see, for example, Weiner, Hospital Practice,pp. 53-58 (Sep. 15, 1995). Additionally, an exemplary list ofbiodegradable polymers and methods of use are described in, e.g.,Handbook of Biodegradable Polymers (Abraham J. Domb et al., eds.,Overseas Publishers Association, 1997); Controlled Drug Delivery:Designing Technologies for the Future (Kinam Park & Randy J. Mrsny eds.,American Chemical Association, 2000); Vernon G. Wong, Method forReducing or Preventing Transplant Rejection in the Eye and IntraocularImplants for Use Therefor, U.S. Pat. No. 6,699,493 (Mar. 2, 2004);Vernon G. Wong & Mae W. L. Hu, Methods for TreatingInflammation-mediated Conditions of the Eye, U.S. Pat. No. 6,726,918(Apr. 27, 2004); David A. Weber et al., Methods and Apparatus forDelivery of Ocular Implants, U.S. Patent Publication No. US2004/0054374(Mar. 18, 2004); Thierry Nivaggioli et al., Biodegradable OcularImplant, U.S. Patent Publication No. US2004/0137059 (Jul. 15, 2004). Ingeneral administration of a tolerogizing composition to an individualcan depend on, e.g., the type immunoresistance, the BoNT/B peptideincluded in the composition, the tolerogizing agent included in thecomposition, and the history, risk factors and symptoms of theindividual.

A tolerogizing composition can be administered to an individual prior toadministering botulinum toxin therapy to prevent the development ofimmunoresistance, during a course of botulinum toxin therapy, or afteronset of immunoresistance, such as, e.g., when symptoms of resistanceare first apparent. In addition, a tolerogizing composition can beadministered to an individual who is at increased risk forimmunoresistance to botulinum toxin therapy. Those skilled in the artwill be able to determine an appropriate candidate for receiving atolerogizing composition of the invention based on, e.g., the particularcondition to be treated and the presence or likelihood of symptoms ofimmunoresistance.

Thus, in one embodiment, a method of the present invention is practicedby administering a tolerogizing composition prior to an individualreceiving a BoNT therapy, such as, e.g., a BoNT/A therapy, a BoNT/Btherapy, a BoNT/C1 therapy, a BoNT/D therapy, a BoNT/E therapy, a BoNT/Ftherapy or a BoNT/G therapy. Such an individual can be, for, e.g., anindividual at increased risk for developing immunoresistance tobotulinum toxin therapy. In another embodiment, a method of the presentinvention is practiced by administering a tolerogizing composition afteran individual has received a BoNT therapy, such as, e.g., a BoNT/Atherapy, a BoNT/B therapy, a BoNT/C1 therapy, a BoNT/D therapy, a BoNT/Etherapy, a BoNT/F therapy or a BoNT/G therapy. In yet anotherembodiment, a method of the present invention is practiced byadministering a tolerogizing composition to an individual who has notbeen diagnosed with a BoNT immunoresistance condition, such as, e.g., aBoNT/A immunoresistance condition, a BoNT/B immunoresistance condition,a BoNT/C1 immunoresistance condition, a BoNT/D immunoresistancecondition, a BoNT/E immunoresistance condition, a BoNT/Fimmunoresistance condition or a BoNT/G immunoresistance condition. Inyet another embodiment, a method of the present invention is practicedby administering a tolerogizing composition to an individual that hasbeen diagnosed with a BoNT/A immunoresistance condition.

In another embodiment, a tolerogizing composition is administered to anindividual. In aspects of this embodiment, a tolerogizing composition isadministered orally to an individual, a tolerogizing composition isadministered topically to an individual, a tolerogizing composition isinjected to an individual or a tolerogizing composition is implanted inan individual.

A tolerogizing composition useful in a method of the invention isadministered to an individual in an effective amount. As used herein,the term “effective amount” when used in reference to treating BoNTimunniresistance means the minimum dose necessary to achieve the desiredtherapeutic effect and includes a dose sufficient to reduce a symptomassociated with a BoNT immunoresistance response. In aspects of thisembodiment, an effect amount of a tolerogizing composition reduces asymptom associated with a BoNT immunoresistance response by, e.g., atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100%. In other aspects of thisembodiment, an effect amount of a tolerogizing composition reduces asymptom associated with a BoNT immunoresistance response by, e.g., atmost 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most80%, at most 90% or at most 100%.

The appropriate effective amount to be administered for a particularapplication of the methods can be determined by those skilled in theart, using the guidance provided herein. For example, an effectiveamount can be extrapolated from in vitro assays and in vivoadministration studies using animal models prior to administration tohumans. Such a effect amount generally is in the range of 0.1-1000mg/day and can be, e.g., in the range of 0.1-500 mg/day, 0.5-500 mg/day,0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or 0.5-5mg/day. An effective dose of a tolerogizing composition useful forinducing tolerance in an individual will depend upon the particularBoNT/B peptide used, the tolerogizing agent used, and the routeadministration. In addition, the actual amount of the effective dose ofa tolerogizing composition to be administered to an individual will bedetermined by a physician taking into account the cause of the BoNTimmunoresistance, the severity of the BoNT immunoresistance and theparticular characteristics of the individual, such as age, weight,general health and the like. Where repeated administration is used, thefrequency of administration depends, in part, on the half-life of thetolerogizing composition. One skilled in the art will recognize that thecondition of the individual can be monitored throughout the course oftherapy and that the effective amount of a tolerogizing composition thatis administered can be adjusted accordingly. It is also understood thatthe frequency and duration of dosing will be dependent, in part, on therelief desired and the half-life of the tolerogizing composition.

Aspects of the present invention provide, in part, a tolerogizingcomposition. It is envisioned that any of the tolerogizing compositiondisclosed in the present specification can be useful in a method oftreating immunoresistance to botulinum toxin therapy in an individual,with the proviso that the tolerogizing composition prevents or reducesthe immunoresistance to a botulinum toxin therapy. Non-limiting examplesinclude tolerogizing compositions comprising BoNT/B peptide derived froma naturally occurring BoNT/B operably linked to a tolerogizing agent,such as, e.g., BoNT/B peptide derived from the BoNT/B of SEQ ID NO: 1operably linked to a tolerogizing agent, a BoNT/B peptide derived from aBoNT/B isoform operably linked to a tolerogizing agent or a BoNT/Bpeptide derived from a BoNT/B subtype operably linked to a tolerogizingagent; and a BoNT/B peptide derived from a non-naturally occurringBoNT/B operably linked to a tolerogizing agent, such as, e.g., a BoNT/Bpeptide derived from a conservative BoNT/B variant operably linked to atolerogizing agent, a BoNT/B peptide derived from a non-conservativeBoNT/B variant operably linked to a tolerogizing agent and a BoNT/Bpeptide derived from a chimeric BoNT/B peptide operably linked to atolerogizing agent. BoNT/B peptides within a tolerogizing compositiondisclosed in the present specification can be selected on, e.g.,immunological factors, such as the selectivity of the BoNT/B peptide foran anti-BoNT antibody, and technical factors, such as chemical synthesisyields. It is also understood that the two or more different BoNT/Bpeptides can be provided separately or as part of a compound moleculesuch as a chimeric BoNT/B peptide.

A tolerogizing composition useful in the invention generally isadministered in a pharmaceutical acceptable composition. As used herein,the term “pharmaceutically acceptable” means any molecular entity orcomposition that does not produce an adverse, allergic or other untowardor unwanted reaction when administered to an individual. As used herein,the term “pharmaceutically acceptable composition” means atherapeutically effective concentration of an active ingredient. Apharmaceutical composition may be administered to a patient alone, or incombination with other supplementary active ingredients, agents, drugsor hormones. The pharmaceutical compositions may be manufactured usingany of a variety of processes, including, without limitation,conventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, and lyophilizing. Thepharmaceutical composition can take any of a variety of forms including,without limitation, a sterile solution, suspension, emulsion,lyophilizate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

It is also envisioned that a pharmaceutical composition disclosed in thepresent specification can optionally include a pharmaceuticallyacceptable carriers that facilitate processing of an active ingredientinto pharmaceutically acceptable compositions. As used herein, the term“pharmacologically acceptable carrier” means any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, auxiliary or excipient.” Such a carriergenerally is mixed with an active compound, or permitted to dilute orenclose the active compound and can be a solid, semi-solid, or liquidagent. It is understood that the active ingredients can be soluble orcan be delivered as a suspension in the desired carrier or diluent. Anyof a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., distilled,deionized water, saline; solvents; dispersion media; coatings;antibacterial and antifungal agents; isotonic and absorption delayingagents; or any other inactive ingredient. Selection of apharmacologically acceptable carrier can depend on the mode ofadministration. Except insofar as any pharmacologically acceptablecarrier is incompatible with the active ingredient, its use inpharmaceutically acceptable compositions is contemplated. Non-limitingexamples of specific uses of such pharmaceutical carriers can be foundin PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C.Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed.1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20 ed. 2000); GOODMAN &GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and HANDBOOK OFPHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003). These protocols are routine procedures and anymodifications are well within the scope of one skilled in the art andfrom the teaching herein.

It is further envisioned that a pharmaceutical composition disclosed inthe present specification can optionally include, without limitation,other pharmaceutically acceptable components, including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE®.Tonicity adjustors useful in a pharmaceutical composition include,without limitation, salts such as, e.g., sodium chloride, potassiumchloride, mannitol or glycerin and other pharmaceutically acceptabletonicity adjustor. The pharmaceutical composition may be provided as asalt and can be formed with many acids, including but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. It is understood that these andother substances known in the art of pharmacology can be included in apharmaceutical composition useful in the invention.

In an embodiment, a method of treating immunoresistance to a botulinumtoxin therapy in an individual comprises the step of administering tothe individual a tolerogizing composition comprising a tolerogizingagent operably linked to a BoNT/B peptide, where the administrationprevents or reduces immunoresistance to botulinum toxin therapy.

Patients treated with a BoNT therapy can develop immunoresistance to thetreatment, thereby reducing or eliminating the beneficial effect of theBoNT therapy. Therefore, blood purifying methods that reduce oreliminate anti-BoNT antibodies from a patient mounting an immuneresponse against a BoNT therapy are of major importance. Immunoaphereticmethods would provide a remedy for BoNT immunoresistance, therebyallowing a patient to continue undergoing a BoNT therapy. Therefore,these methods present a major benefit in terms of providing betterpatient care and reducing health care costs. The BoNT/B peptidesdisclosed in the present specification are useful in methods of reducingor eliminating anti-BoNT antibodies from an individual. In general,blood from an individual exhibiting signs of immunoresistance to a BoNTtherapy can be treated extracorporeally to remove anti-BoNT antibodiesusing an immunosorbent composition comprising at least one BoNT/Bpeptide disclosed in the present specification and the treated bloodreturned back into the individual. Therapeutic immunopheresis has beensuccessfully applied, see, e.g., A. du Moulin et al., Antibody-basedimmunoadsorption as a Therapeutic Means, 11(3) Blood Purif. 145-149(1993); W. O. Richter et al., Efficacy and Safety of ImmunoglobulinApheresis, 43(1) ASAIO J. 53-59 (1997); Watts A. Foley et al., PlasmaPerfusion by Apheresis Through a Gal Immunoaffinity Column SuccessfullyDepletes anti-Gal Antibody: Experience with 320 Aphereses in Baboons, 7Xenotransplant. 181-185 (2000); Monika Graninger et al, ImmunoadsorptionTherapy (Therasorb) in Patients with Severe Lupus Erythematosus, 29Acta. Med. Austriaca 26-29 (2002); Daniel R. Henderson et al., Methodsof Enhancing Effectiveness of Therapeutic Viral Immunogenic AgentAdministration, U.S. Pat. No. 6,406,861 (Jun. 18, 2002); and Robert Kollet al., Treatment of Cardiomyopathy by Removal of Autoantibodies, U.S.Pat. No. 7,022,322 (Apr. 4, 2006).

Thus, the present invention provides, in part, an anti-BoNTimmunoapheresis method of treating immunoresistance to a BoNT therapy inan individual, the method comprising the steps of contacting ananti-BoNT antibody containing component from the individualextracorporeally with a BoNT/B peptide immunosorbent under conditionssuitable for the selective binding of the BoNT/B peptide to theanti-BoNT antibody, the BoNT/B peptide having a length of at least 5amino acids and at most 60 amino acids and returning the anti-BoNTantibody depleted component back into the individual. It is understoodthat any of the above methods of removing botulinum toxin blockingantibodies from a patient can be practiced by selectively removing IgGanti-botulinum toxin antibodies.

Aspects of the present invention provide, in part, an anti-BoNTimmunoapheresis method. As used herein, the term “anti-BoNTimmunoapheresis” is synonymous with “anti-BoNT immunoadsorption” andmeans the separation and removal of anti-BoNT antibodies from ananti-BoNT antibody containing component withdrawn from an individual andthe remainder of the treated anti-BoNT antibody containing componentreturned back into the individual. By definition, anti-BoNT antibodyadsorption by anti-BoNT immunoapheresis is an extracorporeal procedure.

Thus, in an embodiment, an anti-BoNT immunoapheresis method of treatingimmunoresistance to a BoNT therapy in an individual reduces the amountof anti-BoNT antibodies from an anti-BoNT antibody containing component.In aspects of this embodiment, the amount of anti-BoNT antibodiesremoved from an anti-BoNT antibody containing component is, e.g., atleast 10% of the anti-BoNT antibodies from the anti-BoNT antibodycontaining component, at least 20% of the anti-BoNT antibodies from theanti-BoNT antibody containing component, at least 30% of the anti-BoNTantibodies from the anti-BoNT antibody containing component, at least40% of the anti-BoNT antibodies from the anti-BoNT antibody containingcomponent, at least 50% of the anti-BoNT antibodies from the anti-BoNTantibody containing component, at least 60% of the anti-BoNT antibodiesfrom the anti-BoNT antibody containing component, at least 70% of theanti-BoNT antibodies from the anti-BoNT antibody containing component,at least 80% of the anti-BoNT antibodies from the anti-BoNT antibodycontaining component and at least 90% of the anti-BoNT antibodies fromthe anti-BoNT antibody containing component.

In other aspects of this embodiment, the amount of anti-BoNT antibodiesremoved from an anti-BoNT antibody containing component is, e.g., atmost 10% of the anti-BoNT antibodies from the anti-BoNT antibodycontaining component, at most 20% of the anti-BoNT antibodies from theanti-BoNT antibody containing component, at most 30% of the anti-BoNTantibodies from the anti-BoNT antibody containing component, at most 40%of the anti-BoNT antibodies from the anti-BoNT antibody containingcomponent, at most 50% of the anti-BoNT antibodies from the anti-BoNTantibody containing component, at most 60% of the anti-BoNT antibodiesfrom the anti-BoNT antibody containing component, at most 70% of theanti-BoNT antibodies from the anti-BoNT antibody containing component,at most 80% of the anti-BoNT antibodies from the anti-BoNT antibodycontaining component and at most 90% of the anti-BoNT antibodies fromthe anti-BoNT antibody containing component.

Aspects of the present invention provide, in part, an anti-BoNT antibodycontaining component from the individual. Non-limiting examples of ananti-BoNT antibody containing component from the individual include,blood, serum, an isolated IgG antibody component and lymph fluid.Typically, blood removal and serum separation are achieved using anautomated blood cell separator machine, see, e.g., Alessandro Zuccatoand Rigaste S. Zeno, Method for the Specific Immunoadsorption ofSelected Pathogenic Factors, PCT Publication WO 96/16666 (Jun. 6, 1996);and Robert Koll et al., Treatment of Cardiomyopathy by Removal ofAutoantibodies, U.S. Pat. No. 7,022,322 (Apr. 4, 2006). Non-limitingexamples of such a machine include, e.g., an autopheresis-CtmTherapeutic Plasma System (TPS) is employed (Baxter Healthcare Corp,Deerfield, Ill.) and a COBE-Spectra pheresis unit (Blood ComponentTechnology, Inc., Lakewood, Col.).

Aspects of the present invention provide, in part, a BoNT/B peptideimmunosorbent. As used herein, the term “BoNT/B peptide immunosorbent”means a molecule comprising a BoNT/B peptide that selectively binds toan anti-BoNT antibody. It is envisioned that any of the BoNT/B peptidesdisclosed in the present specification can be useful for anti-BoNTimmunoapheresis for extracorporeal removal of anti-BoNT antibodies.Non-limiting examples include a BoNT/B peptide derived from a naturallyoccurring BoNT/B, such as, e.g., the BoNT/B of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO:13, a BoNT/B isoform or a BoNT/B subtype; and a BoNT/B peptide derivedfrom a non-naturally occurring BoNT/B, such as, e.g., a conservativeBoNT/B variant, a non-conservative BoNT/B variant and a chimeric BoNT/Bpeptide. BoNT/B peptides disclosed in the present specification can beselected, e.g., depending on immunological factors, such as potency ofthe peptide in eliciting an immunogenic response, and technical factors,such as chemical synthesis yields. It is also understood that the two ormore different BoNT/B peptides can be provided separately or as part ofa compound molecule such as a chimeric BoNT/B peptide.

Thus, in an embodiment, a BoNT/B peptide immunosorbent comprises aBoNT/B peptide having a length of at least 5 amino acids and at most 60amino acids. In aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises a BoNT/B peptide of SEQ ID NO: 1 having a lengthof at least 5 amino acids and at most 60 amino acids, a BoNT/B peptideof SEQ ID NO: 3 having a length of at least 5 amino acids and at most 60amino acids, a BoNT/B peptide of SEQ ID NO: 5 having a length of atleast 5 amino acids and at most 60 amino acids, a BoNT/B peptide of SEQID NO: 7 having a length of at least 5 amino acids and at most 60 aminoacids, a BoNT/B peptide of SEQ ID NO: 9 having a length of at least 5amino acids and at most 60 amino acids, a BoNT/B peptide of SEQ ID NO:11 having a length of at least 5 amino acids and at most 60 amino acidsor a BoNT/B peptide of SEQ ID NO: 11 having a length of at least 5 aminoacids and at most 60 amino acids.

In other aspects of this embodiment, a BoNT/B peptide immunosorbentcomprises a BoNT/B peptide comprising an amino acid sequence selectedfrom the group consisting of amino acids 610-628 of SEQ ID NO: 1, aminoacids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5,amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO:5, amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ IDNO: 3, amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQID NO: 7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 ofSEQ ID NO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids890-908 of SEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, aminoacids 918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1,amino acids 932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO:1, amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ IDNO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 ofSEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, aminoacids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3,amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ IDNO: 7, amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 ofSEQ ID NO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3,amino acids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQID NO: 7.

In other aspects of this embodiment, a BoNT/B peptide immunosorbentcomprises a BoNT/B peptide comprising an amino acid sequence selectedfrom the group consisting of amino acids 736-754 of SEQ ID NO: 1, aminoacids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1,amino acids 778-796 of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO:1, amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ IDNO: 5, amino acids 890-908 of SEQ ID NO: 7, amino acids 932-950 of SEQID NO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids 974-992 ofSEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, aminoacids 1058-1076 of SEQ ID NO: 7, amino acids 1268-1291 of SEQ ID NO: 1,amino acids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ IDNO: 5 and amino acids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises a BoNT/B peptide comprising an amino acidsequence selected from the group consisting of amino acids 616-626 ofSEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, amino acids 735-745of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1, amino acids867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 7, aminoacids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ ID NO: 3,amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQ ID NO:1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 of SEQ IDNO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids 1039-1049 ofSEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, amino acids1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3, aminoacids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ ID NO: 7,amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 of SEQ IDNO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids 1269-1281 ofSEQ ID NO: 7.

In another embodiment, a BoNT/B peptide immunosorbent comprises a BoNT/Bpeptide having a length of at least 5 amino acids and at most 60 aminoacids and is derived from a naturally occurring BoNT/B. In aspects ofthis embodiment, the naturally occurring BoNT/B is a BoNT/B isoform or aBoNT/B subtype.

In another embodiment, a BoNT/B peptide immunosorbent comprises a BoNT/Bpeptide having a length of at least 5 amino acids and at most 60 aminoacids and is derived from a non-naturally occurring BoNT/B. In aspectsof this embodiment, the naturally occurring BoNT/B is a conservativeBoNT/B variant or a non-conservative BoNT/B variant.

In aspects of this embodiment, a BoNT/B peptide immunosorbent comprisesa BoNT/B peptide comprising, e.g., 1-4 conservative amino acidsubstitutions to amino acids 610-628 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 736-754 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 736-754 of SEQ IDNO: 5, 1-4 conservative amino acid substitutions to amino acids 778-796of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 778-796 of SEQ ID NO: 5, 1-4 conservative amino acid substitutionsto amino acids 820-838 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 820-838 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 820-838 of SEQ ID NO: 5, 1-4conservative amino acid substitutions to amino acids 820-838 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids 862-880of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 862-880 of SEQ ID NO: 7, 1-4 conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 5, 1-4conservative amino acid substitutions to amino acids 890-908 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids 918-936of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 918-936 of SEQ ID NO: 3, 1-4 conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 932-950 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 960-978 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 960-978 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids 960-978of SEQ ID NO: 7, 1-4 conservative amino acid substitutions to aminoacids 974-992 of SEQ ID NO: 1, 1-4 conservative amino acid substitutionsto amino acids 974-992 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 1030-1048 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1030-1048 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 5, 1-4 conservative amino acid substitutions toamino acids 1030-1048 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 5, 1-4 conservative amino acid substitutions to amino acids1058-1076 of SEQ ID NO: 7, 1-4 conservative amino acid substitutions toamino acids 1072-1090 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 1072-1090 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 1072-1090 of SEQ ID NO: 7, 1-4conservative amino acid substitutions to amino acids 1254-1272 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids1254-1272 of SEQ ID NO: 3, 1-4 conservative amino acid substitutions toamino acids 1268-1291 of SEQ ID NO: 1, 1-4 conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 3, 1-4 conservativeamino acid substitutions to amino acids 1268-1291 of SEQ ID NO: 5 or 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 7.

In other aspects of this embodiment, a BoNT/B peptide immunosorbentcomprises a BoNT/B peptide comprising, e.g., 1-4 conservative amino acidsubstitutions to amino acids 736-754 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 736-754 of SEQ ID NO: 5, 1-4conservative amino acid substitutions to amino acids 778-796 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids 778-796of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 890-908 of SEQ ID NO: 1, 1-4 conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 890-908 of SEQ ID NO: 7, 1-4conservative amino acid substitutions to amino acids 932-950 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids 932-950of SEQ ID NO: 5, 1-4 conservative amino acid substitutions to aminoacids 974-992 of SEQ ID NO: 1, 1-4 conservative amino acid substitutionsto amino acids 974-992 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 7, 1-4 conservativeamino acid substitutions to amino acids 1058-1076 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 1058-1076 of SEQ IDNO: 3, 1-4 conservative amino acid substitutions to amino acids1058-1076 of SEQ ID NO: 5, 1-4 conservative amino acid substitutions toamino acids 1058-1076 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 1268-1291 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 1268-1291 of SEQ IDNO: 5 or 1-4 conservative amino acid substitutions to amino acids1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/B peptide immunosorbentcomprises a BoNT/B peptide comprising, e.g., 1-4 conservative amino acidsubstitutions to amino acids 616-626 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 735-745 of SEQ ID NO: 1, 1-4conservative amino acid substitutions to amino acids 735-745 of SEQ IDNO: 5, 1-4 conservative amino acid substitutions to amino acids 778-789of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 867-877 of SEQ ID NO: 1, 1-4 conservative amino acid substitutionsto amino acids 867-877 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 895-905 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 895-905 of SEQ ID NO: 3, 1-4conservative amino acid substitutions to amino acids 929-939 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids 974-984of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to aminoacids 974-984 of SEQ ID NO: 3, 1-4 conservative amino acid substitutionsto amino acids 974-984 of SEQ ID NO: 7, 1-4 conservative amino acidsubstitutions to amino acids 1039-1049 of SEQ ID NO: 1, 1-4 conservativeamino acid substitutions to amino acids 1039-1049 of SEQ ID NO: 5, 1-4conservative amino acid substitutions to amino acids 1039-1049 of SEQ IDNO: 7, 1-4 conservative amino acid substitutions to amino acids1065-1075 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions toamino acids 1065-1075 of SEQ ID NO: 3, 1-4 conservative amino acidsubstitutions to amino acids 1065-1075 of SEQ ID NO: 5, 1-4 conservativeamino acid substitutions to amino acids 1065-1075 of SEQ ID NO: 7, 1-4conservative amino acid substitutions to amino acids 1269-1281 of SEQ IDNO: 1, 1-4 conservative amino acid substitutions to amino acids1269-1281 of SEQ ID NO: 3, 1-4 conservative amino acid substitutions toamino acids 1269-1281 of SEQ ID NO: 5 or 1-4 conservative amino acidsubstitutions to amino acids 1269-1281 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises a BoNT/B peptide comprising, e.g., 1-4non-conservative amino acid substitutions to amino acids 610-628 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 736-754 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 778-796 of SEQID NO: 5, 1-4 non-conservative amino acid substitutions to amino acids820-838 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 820-838 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 820-838 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 820-838 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids862-880 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 862-880 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 5, 1-4 non-conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 918-936 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 918-936 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 932-950 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 960-978 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 960-978 of SEQID NO: 3, 1-4 non-conservative amino acid substitutions to amino acids960-978 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 974-992 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids1030-1048 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 1030-1048 of SEQ ID NO: 3, 1-4 non-conservative aminoacid substitutions to amino acids 1030-1048 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 1030-1048 ofSEQ ID NO: 7, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1058-1076 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 5, 1-4 non-conservative amino acid substitutions to aminoacids 1058-1076 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1072-1090 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1072-1090 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1072-1090 of SEQ ID NO: 7, 1-4 non-conservative amino acidsubstitutions to amino acids 1254-1272 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 1254-1272 ofSEQ ID NO: 3, 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 5 or 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises a BoNT/B peptide comprising, e.g., 1-4non-conservative amino acid substitutions to amino acids 736-754 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids736-754 of SEQ ID NO: 5, 1-4 non-conservative amino acid substitutionsto amino acids 778-796 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 778-796 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 890-908 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids890-908 of SEQ ID NO: 3, 1-4 non-conservative amino acid substitutionsto amino acids 890-908 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 890-908 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 932-950 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids932-950 of SEQ ID NO: 5, 1-4 non-conservative amino acid substitutionsto amino acids 974-992 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 974-992 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 974-992 of SEQID NO: 7, 1-4 non-conservative amino acid substitutions to amino acids1058-1076 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 1058-1076 of SEQ ID NO: 3, 1-4 non-conservative aminoacid substitutions to amino acids 1058-1076 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 1058-1076 ofSEQ ID NO: 7, 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1268-1291 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1268-1291 ofSEQ ID NO: 5 or 1-4 non-conservative amino acid substitutions to aminoacids 1268-1291 of SEQ ID NO: 7.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises a BoNT/B peptide comprising, e.g., 1-4non-conservative amino acid substitutions to amino acids 616-626 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids735-745 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 735-745 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 778-789 of SEQ ID NO: 1, 1-4non-conservative amino acid substitutions to amino acids 867-877 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids867-877 of SEQ ID NO: 7, 1-4 non-conservative amino acid substitutionsto amino acids 895-905 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 895-905 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 929-939 of SEQID NO: 1, 1-4 non-conservative amino acid substitutions to amino acids974-984 of SEQ ID NO: 1, 1-4 non-conservative amino acid substitutionsto amino acids 974-984 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 974-984 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 1039-1049 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 1039-1049 of SEQ ID NO: 5, 1-4 non-conservative amino acidsubstitutions to amino acids 1039-1049 of SEQ ID NO: 7, 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 1, 1-4 non-conservative amino acid substitutions to aminoacids 1065-1075 of SEQ ID NO: 3, 1-4 non-conservative amino acidsubstitutions to amino acids 1065-1075 of SEQ ID NO: 5, 1-4non-conservative amino acid substitutions to amino acids 1065-1075 ofSEQ ID NO: 7, 1-4 non-conservative amino acid substitutions to aminoacids 1269-1281 of SEQ ID NO: 1, 1-4 non-conservative amino acidsubstitutions to amino acids 1269-1281 of SEQ ID NO: 3, 1-4non-conservative amino acid substitutions to amino acids 1269-1281 ofSEQ ID NO: 5 or 1-4 non-conservative amino acid substitutions to aminoacids 1269-1281 of SEQ ID NO: 7.

In another embodiment, a BoNT/B peptide immunosorbent comprises animmunoreactive fragment of a BoNT/B peptide having a length of at leastfive amino acids and at most 60 amino acids. In aspects of thisembodiment, a BoNT/B peptide immunosorbent comprises an immunoreactivefragment of a BoNT/B peptide comprising, e.g., at least 6 consecutiveamino acids, at least 7 consecutive amino acids, at least 8 consecutiveamino acids, at least 9 consecutive amino acids, at least 10 consecutiveamino acids, at least 12 consecutive amino acids, at least 15consecutive amino acids, at least 18 consecutive amino acids or at least20 consecutive amino acids. In other aspects of this embodiment, aBoNT/B peptide immunosorbent comprises an immunoreactive fragment of aBoNT/B peptide comprising, e.g., at most 6 consecutive amino acids, atmost 7 consecutive amino acids, at most 8 consecutive amino acids, atmost 9 consecutive amino acids, at most 10 consecutive amino acids, atmost 12 consecutive amino acids, at most 15 consecutive amino acids, atmost 18 consecutive amino acids or at most 20 consecutive amino acids.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises an immunoreactive fragment of a BoNT/B peptidecomprising, e.g., at least 6 non-consecutive amino acids, at least 7non-consecutive amino acids, at least 8 non-consecutive amino acids, atleast 9 non-consecutive amino acids, at least 10 non-consecutive aminoacids, at least 12 non-consecutive amino acids, at least 15non-consecutive amino acids, at least 18 non-consecutive amino acids orat least 20 non-consecutive amino acids. In still other aspects of thisembodiment, a BoNT/B peptide immunosorbent comprises an immunoreactivefragment of a BoNT/B peptide comprising, e.g., at most 6 non-consecutiveamino acids, at most 7 non-consecutive amino acids, at most 8non-consecutive amino acids, at most 9 non-consecutive amino acids, atmost 10 non-consecutive amino acids, at most 12 non-consecutive aminoacids, at most 15 non-consecutive amino acids, at most 18non-consecutive amino acids or at most 20 non-consecutive amino acids.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises an immunoreactive fragment of a BoNT/B peptidecomprising having from five to sixty amino acids, from five to fiftyamino acids, from eight to fifty amino acids, from ten to fifty aminoacids, from five to twenty amino acids, from eight to twenty aminoacids, from ten to twenty amino acids, from twelve to twenty amino acidsor from fifteen to twenty amino acids.

In another embodiment, a BoNT/B peptide comprises an immunogenic BoNT/Bfragment. In an aspect of this embodiment, an immunogenic BoNT/Bfragment comprises at least six consecutive amino acids of a BoNT/Bpeptide. In other aspects of this embodiment, an immunogenic BoNT/Bfragment comprises at least six consecutive amino acids from, e.g.,amino acids 610-628 of SEQ ID NO: 1

In another embodiment, a BoNT/B peptide immunosorbent comprises animmunoreactive BoNT/B fragment. In an aspect of this embodiment, aBoNT/B peptide immunosorbent comprises an immunoreactive BoNT/Bfragment, the immunoreactive BoNT/B fragment comprising at least sixconsecutive amino acids of a BoNT/B peptide. In other aspects of thisembodiment, a BoNT/B peptide immunosorbent comprises an immunoreactiveBoNT/B fragment, the immunoreactive BoNT/B fragment comprising at leastsix consecutive amino acids from, e.g., amino acids 610-628 of SEQ IDNO: 1, amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQID NO: 5, amino acids 778-796 of SEQ ID NO: 1, amino acids 778-796 ofSEQ ID NO: 5, amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838of SEQ ID NO: 3, amino acids 820-838 of SEQ ID NO: 5, amino acids820-838 of SEQ ID NO: 7, amino acids 862-880 of SEQ ID NO: 1, aminoacids 862-880 of SEQ ID NO: 7, amino acids 890-908 of SEQ ID NO: 1,amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908 of SEQ ID NO:5, amino acids 890-908 of SEQ ID NO: 7, amino acids 918-936 of SEQ IDNO: 1, amino acids 918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQID NO: 1, amino acids 932-950 of SEQ ID NO: 5, amino acids 960-978 ofSEQ ID NO: 1, amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, aminoacids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3,amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ IDNO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 ofSEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 of SEQ ID NO: 1, aminoacids 1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 of SEQ ID NO: 7,amino acids 1254-1272 of SEQ ID NO: 1, amino acids 1254-1272 of SEQ IDNO: 3, amino acids 1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 ofSEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5 and amino acids1268-1291 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/B peptide immunosorbentcomprises an immunoreactive BoNT/B fragment, the immunoreactive BoNT/Bfragment comprising at least six consecutive amino acids from, e.g.,amino acids 616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO:1, amino acids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ IDNO: 1, amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQID NO: 7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 ofSEQ ID NO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984of SEQ ID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids974-984 of SEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, aminoacids 1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7,amino acids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ IDNO: 3, amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 ofSEQ ID NO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 oramino acids 1269-1281 of SEQ ID NO: 7.

In another aspect of this embodiment, a BoNT/B peptide immunosorbentcomprises an immunoreactive BoNT/B fragment, the immunoreactive BoNT/Bfragment comprising at least six non-consecutive amino acids of a BoNT/Bpeptide. In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises an immunoreactive BoNT/B fragment, theimmunoreactive BoNT/B fragment comprising at least six non-consecutiveamino acids from, e.g., amino acids 610-628 of SEQ ID NO: 1, amino acids736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, aminoacids 778-796 of SEQ ID NO: 1, amino acids 778-796 of SEQ ID NO: 5,amino acids 820-838 of SEQ ID NO: 1, amino acids 820-838 of SEQ ID NO:3, amino acids 820-838 of SEQ ID NO: 5, amino acids 820-838 of SEQ IDNO: 7, amino acids 862-880 of SEQ ID NO: 1, amino acids 862-880 of SEQID NO: 7, amino acids 890-908 of SEQ ID NO: 1, amino acids 890-908 ofSEQ ID NO: 3, amino acids 890-908 of SEQ ID NO: 5, amino acids 890-908of SEQ ID NO: 7, amino acids 918-936 of SEQ ID NO: 1, amino acids918-936 of SEQ ID NO: 3, amino acids 932-950 of SEQ ID NO: 1, aminoacids 932-950 of SEQ ID NO: 5, amino acids 960-978 of SEQ ID NO: 1,amino acids 960-978 of SEQ ID NO: 3, amino acids 960-978 of SEQ ID NO:7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ IDNO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, aminoacids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7,amino acids 1072-1090 of SEQ ID NO: 1, amino acids 1072-1090 of SEQ IDNO: 3, amino acids 1072-1090 of SEQ ID NO: 7, amino acids 1254-1272 ofSEQ ID NO: 1, amino acids 1254-1272 of SEQ ID NO: 3, amino acids1268-1291 of SEQ ID NO: 1, amino acids 1268-1291 of SEQ ID NO: 3, aminoacids 1268-1291 of SEQ ID NO: 5 and amino acids 1268-1291 of SEQ ID NO:7.

In still other aspects of this embodiment, a BoNT/B peptideimmunosorbent comprises an immunoreactive BoNT/B fragment, theimmunoreactive BoNT/B fragment comprising at least six non-consecutiveamino acids from, e.g., amino acids 616-626 of SEQ ID NO: 1, amino acids735-745 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 5, aminoacids 778-789 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 7, amino acids 895-905 of SEQ ID NO:1, amino acids 895-905 of SEQ ID NO: 3, amino acids 929-939 of SEQ IDNO: 1, amino acids 974-984 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 3, amino acids 974-984 of SEQ ID NO: 7, amino acids 1039-1049 ofSEQ ID NO: 1, amino acids 1039-1049 of SEQ ID NO: 5, amino acids1039-1049 of SEQ ID NO: 7, amino acids 1065-1075 of SEQ ID NO: 1, aminoacids 1065-1075 of SEQ ID NO: 3, amino acids 1065-1075 of SEQ ID NO: 5,amino acids 1065-1075 of SEQ ID NO: 7, amino acids 1269-1281 of SEQ IDNO: 1, amino acids 1269-1281 of SEQ ID NO: 3, amino acids 1269-1281 ofSEQ ID NO: 5 or amino acids 1269-1281 of SEQ ID NO: 7.

Aspects of an anti-BoNT immunoapheresis method disclosed in the presentspecification can be practiced using a solid support. As a non-limitingexample, a solid phase system can utilize a solid phase matrix which isa solid phrase support comprising one or more a BoNT/B peptideimmunosorbents. The blood, plasma or serum containing the anti-BoNTantibodies is passed over the solid support, exiting the solid supportand leaving behind the anti-BoNT antibody/BoNT/B peptide complexes. Suchsolid supports are chemically inert with respect to antibody-containingfluids, have sufficient binding capacity, and generally are in the formof a continuous large surface such as a sheet or column, or in the formof particles or vesicles. Exemplary solid supports useful in theinvention, including those useful for affinity chromatography,encompass, without limitation, silica; synthetic silicates, such as,e.g., porous glass, like glass fiber filters; biogenic silicates, suchas, e.g., diatomaceous earth; silicate-containing materials, such as,e.g., kaolinite and borosilicate; and synthetic polymers, such as, e.g.,polystyrene, polypropylene and polysaccharides, see, e.g., A. HeatherGood, et al., Methods and Compositions for Attenuating Antibody-mediatedXenograft Rejection in Human Recipients, U.S. Pat. No. 6,607,723 (Aug.19, 2003); and Mazid, supra, 1992. An affinity column is a cylindricalcontainer with filters on both ends which contains a solid support towhich the one or more BoNT/B peptides are bound. One skilled in the artunderstands that plasma or serum generally is passed through a columnsince whole blood contains cells and particulate matter such asplatelets which can impede column flow.

Solid supports useful in aspects of the invention further include, yetare not limited to, agarose, which is a neutral linear polysaccharidegenerally composed of D-galactose and altered 3,6-anhydrogalactoseresidues, for example, Sepharose; activated gels, cellulose,nitrocellulose, polyvinylchloride, and diazotized paper. The skilledperson understands that these and a variety of other well known solidsupports can be useful in the methods of the invention.

BoNT/B peptides can be covalently or noncovalently bound to the solidsupport using well known methods. Supports which can be non-covalentlybound by incubation with the immunosorbent include, without limitation,a nitrocellulose support, a borosilicate support, a polyvinylchloridesupport, a polystyrene support and a diazotized support. Activated solidsupports such as activated matrices also are well known in the art andcommercially available and useful in the invention. Such activated solidsupports encompass, without limitation, an epoxy-activated agarosesupport, such as, e.g., a CNBr-activated agarose support, a6-aminohexanoic acid support and a 1,6-diaminohexane-agarose support; athiopropyl agarose support; a carbonyldiimidazole-activated agarosesupport; and an aminoethyl support and a hydrazide-activatedpolyacrylamide support, see, e.g., Daniel R. Henderson et al., Methodsof Enhancing Effectiveness of Therapeutic Viral Immunogenic AgentAdministration, U.S. Pat. No. 6,406,861 (Jun. 18, 2001; and Joseph P.Balint, Anti-human IGM Immunoadsorbent and Process for Producing SaidImmunoadsorbent, U.S. Pat. No. 4,762,787 (Aug. 9, 1988).

In one embodiment, an anti-BoNT immunoapheresis method disclosed in thepresent specification can be practiced using a solid support comprisingone or more a BoNT/B peptide immunosorbents. In an aspect of thisembodiment, an anti-BoNT immunoapheresis method disclosed in the presentspecification can be practiced using an affinity column comprising oneor more a BoNT/B peptide immunosorbents.

In other aspects of this embodiment, an anti-BoNT immunoapheresis methoddisclosed in the present specification can be practiced using a silicasupport comprising one or more a BoNT/B peptide immunosorbents, asynthetic silicate support comprising one or more a BoNT/B peptideimmunosorbents, a biogenic silicate support comprising one or more aBoNT/B peptide immunosorbents and a synthetic polymer support comprisingone or more a BoNT/B peptide immunosorbents.

In other aspects of this embodiment, an anti-BoNT immunoapheresis methoddisclosed in the present specification can be practiced using aSepharose support comprising one or more a BoNT/B peptideimmunosorbents, a polyvinylchloride support comprising one or more aBoNT/B peptide immunosorbents, a cellulose support comprising one ormore a BoNT/B peptide immunosorbents, a nitrocellulose supportcomprising one or more a BoNT/B peptide immunosorbents, a borosilicatesupport comprising one or more a BoNT/B peptide immunosorbents, apolyvinylchloride support comprising one or more a BoNT/B peptideimmunosorbents, a polystyrene support comprising one or more a BoNT/Bpeptide immunosorbents, a polypropylene support comprising one or more aBoNT/B peptide immunosorbents and a diazotized support comprising one ormore a BoNT/B peptide immunosorbents.

In yet other aspects of this embodiment, an anti-BoNT immunoapheresismethod disclosed in the present specification can be practiced using anepoxy-activated agarose support comprising one or more a BoNT/B peptideimmunosorbents, a thiopropyl agarose support comprising one or more aBoNT/B peptide immunosorbents, a carbonyldiimidazole-activated agarosesupport comprising one or more a BoNT/B peptide immunosorbents, anaminoethyl support comprising one or more a BoNT/B peptideimmunosorbents and a hydrazide-activated polyacrylamide supportcomprising one or more a BoNT/B peptide immunosorbents. In other aspectsof this embodiment, an anti-BoNT immunoapheresis method disclosed in thepresent specification can be practiced using a CNBr-activated agarosesupport comprising one or more a BoNT/B peptide immunosorbents, a6-aminohexanoic acid support comprising one or more a BoNT/B peptideimmunosorbents and a 1,6-diaminohexane-agarose support comprising one ormore a BoNT/B peptide immunosorbents.

Aspects of an anti-BoNT immunoapheresis method disclosed in the presentspecification can be practiced using liquid phase separation. Liquidphase separation can be performed, e.g., by conjugating one or moreBoNT/B peptide immunosorbents to a hapten such as, without limitation,dinitrophenol or fluorescein. After mixing the hapten/BoNT/B peptideconjugate with an individual's blood, plasma or serum, the BoNT/Bpeptide conjugate forms complexes with anti-BoNT antibodies. As anon-limiting example, such antibody-peptide complexes can beprecipitated using polyethylene glycol (PEG), and the precipitatedcomplexes separated from the blood, plasma or serum usingcentrifugation, see, e.g., Paul A. Liberti & Paul Pollara, SelectiveRemoval of Immunospecifically Recognizable Substances from Solution,U.S. Pat. No. 4,551,435 (Nov. 5, 1985). One skilled in the artappreciates that these and other solid-phase and liquid-phase systemscan be used to separate anti-BoNT antibody-BoNT/B peptide complexes froman individual's blood, plasma or serum.

In an embodiment, an anti-BoNT immunoapheresis method disclosed in thepresent specification can be practiced using liquid phase separationcomprising one or more a BoNT/B peptide immunosorbents. In an aspect ofthis embodiment, an anti-BoNT immunoapheresis method disclosed in thepresent specification can be practiced using liquid phase separationcomprising one or more a BoNT/B peptide immunosorbents conjugated tohapten. In other aspects of this embodiment, an anti-BoNTimmunoapheresis method disclosed in the present specification can bepracticed using liquid phase separation comprising one or more a BoNT/Bpeptide immunosorbents conjugated to dinitrophenol or fluorescein.

In still a further embodiment, one or more BoNT/B peptides are bound tolipid vesicles, and the lipid vesicle-immunosorbent is mixed with apatient's plasma or serum to allow binding to the blocking antibodies.The plasma or serum is subsequently filtered to remove the lipidvesicle-immunosorbent-antibody complex, see, e.g., James F. Marten,Therapeutic Apheresis, U.S. Pat. No. 4,643,718 (Feb. 17, 1987).

It is understood that the blood, serum, plasma or lymph are contactedwith the one or more BoNT/B peptides attached to a solid support underconditions that promote binding between the one or more BoNT/B peptidesand anti-BoNT antibodies, see, e.g., M. Abdul Mazid, Affinity Supportsfor Hemoperfusion, U.S. Pat. No. 5,149,425 (Sep. 22, 1992). It is alsounderstood that the blood, serum, plasma or lymph can be contacted witha sequential series of a different BoNT/B peptides, such as, e.g., twoor more solid supports each comprising a different BoNT/B peptide. Suchconditions can include, without limitation, contact temperatures in therange of 35° C. and 40° C., and contact times of about one to six hours.

Aspects of the present invention provide, in part, returning theanti-BoNT antibody-depleted component back into the individual. It isunderstood that the unbound portion of the blood, plasma, or serum,which is significantly anti-BoNT antibody-depleted, is reintegrated withcellular components of blood as necessary and reintroduced into theindividual on a continuous basis or following collection. It is alsounderstood that the blood can be pre-warmed to body temperature beforethe blood is returned to the individual. One skilled in the art furtherunderstands that, if desired, the antibody-depleted blood, plasma orserum can be assayed prior to reintroduction in the individual, e.g.,using one of the BoNT/B peptide binding assays or protection assaysdisclosed herein.

It is further understood that pre-clearance of antibodies, or a class ofantibody such as the IgG class, can be performed prior to selectiveremoval of anti-BoNT antibodies. From the pre-cleared antibody pool,BoNT/B peptide-reactive antibodies can be removed, and the remainingantibodies from the pre-cleared pool reconstituted into the blood andreperfused into the individual, thus reducing the volume to be passedover the blocking antibody selective support and also reducingnon-specific binding. As a non-limiting example, non-specific Protein GSepharose columns such as PROSORBA® (IMRE; Munich, Germany) orIg-THERASORB® (Plasmaselect; Teterow, Germany) can be used to remove asignificant portion of IgG antibody. A variety of additional techniquessuitable for general pre-clearance of antibodies are well known in theart and include, yet are not limited to, ammonium sulfate precipitationwith ion exchange chromatography; caprylic acid; DEAE-matrices(ion-exchange chromatography); hydroxyapatite chromatography, and gelfiltration (Sepharose), see, e.g., Harlow & Lane, supra, 1998a; andHarlow & Lane, supra, 1998b.

The present invention provides, in part, a method of inducing a BoNT/Bimmune response in an individual, the method comprising the step ofadministering to the individual an immune response inducing compositioncomprising an adjuvant and a BoNT/B antigen, where administration of theimmune response inducing composition produces an immune response in theindividual.

The present invention provides, in part, a method of producinganti-BoNT/B antibodies in an individual, the method comprising the stepsof administering to the individual an immune response inducingcomposition comprising an adjuvant and a BoNT/B antigen, whereadministration of the immune response inducing composition produces animmune response in the individual; collecting from the individual asample containing the anti-BoNT/B antibody or anti-BoNT/Bantibody-producing cell; and isolating the anti-BoNT/B antibody from thesample. Antibodies to be prepared according to a method of the inventioninclude polyclonal and monoclonal antibodies. Anti-BoNT/B polyclonalantibodies or a monoclonal anti-BoNT/B antibody can be used in a varietyof applications, including, without limitation, detection of botulinumtoxin in a sample, such as, e.g., a substance suspected to becontaminated with a BoNT/B.

Aspects of the present invention provide, in part, an immune responseinducing composition comprising a BoNT/B antigen and an adjuvant capableproducing an immune response. It is envisioned that any of the BoNT/Bantigens disclosed in the present specification can be useful in animmune response inducing composition, with the proviso that the BoNT/Bantigen induces a specific immunological response to a BoNT/B.Non-limiting examples include a BoNT/B antigen derived from a naturallyoccurring BoNT/B, such as, e.g., the BoNT/B of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO:13, a BoNT/B isoform or a BoNT/B subtype; and a BoNT/B antigen derivedfrom a non-naturally occurring BoNT/B, such as, e.g., a conservativeBoNT/B variant, a non-conservative BoNT/B variant and a chimeric BoNT/Bantigen. BoNT/B peptides disclosed in the present specification can beselected, e.g., depending on immunological factors, such as potency ofthe peptide in inducing an immune response, and technical factors, suchas chemical synthesis yields. It is also understood that the two or moredifferent BoNT/B antigen can be provided separately or as part of acompound molecule such as a chimeric BoNT/B antigen.

It is envisioned that any and all adjuvants can be useful in such animmune response inducing composition. As used herein, the term“adjuvant” when used in reference to an immune response inducingcomposition means any substance or mixture of substances that increasesor diversifies the immune response to an antigenic compound. An adjuvantcan, for example, serve to reduce the number of immunizations or theamount of antigen required for protective immunization. The use ofadjuvants in an immune response inducing composition is well known. Themain objective of these adjuvants is to allow an increase in the immuneresponse. These adjuvants are diverse in nature. Various adjuvants usedto increase the immunological response include, but are not limited to,e.g., the Freund type of adjuvants, such as, e.g., Freund's completeadjuvant (FCA); Freund's incomplete adjuvant (FIA); sapogeninglycosides, such as, e.g., saponins; carbopol;N-acetylmuramyl-L-alanyl-D-isoglutamine (commonly known as muramyldipeptide or “MDP”); lipopolysaccharide (LPS), surface activesubstances, such as, e.g., lysolecithin, pluronic polyols, polyanions,peptides and dinitrophenol; adjuvants usable in humans such as BacilleCalmette-Guerin and Corynebacterium parvum, or similar immunostimulatoryagents. Additional examples of adjuvants which can be employed includeMPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). Such adjuvants are generally used in the form of anemulsion with an aqueous phase, or, more commonly, may consist ofwater-insoluble inorganic salts. These inorganic salts may consist, forexample, of aluminum hydroxide, zinc sulfate, colloidal iron hydroxide,calcium phosphate or calcium chloride. Aluminum hydroxide (Al(OH)₃) is acommonly used adjuvant. Currently, the only FDA-approved adjuvant foruse in humans is aluminum salts (Alum) which are used to “depot”antigens by precipitation of the antigens. Adjuvants provided above aremerely exemplary. In fact, any adjuvant may be used in the immunogeniccomposition of the present invention as long as the adjuvant satisfiesthe requisite characteristics that are necessary for practicing thepresent invention. As indicated above, the carrier of the compositionsof the present invention itself may act as an adjuvant. Specificadjuvants and methods of making and using are described in, e.g., Guptaet al. Vaccine, 11: 993-306, 1993; Arnon, R. (Ed.) Synthetic Vaccines1:83-92, CRC Press, Inc., Boca Raton, Fla., 1987; and David W. Waggoner,Jr. et al., Immunogenicity-Enhancing Carriers and Compositions Thereofand Methods of Using the Same, U.S. Patent Publication No. 20040057958(Mar. 25, 2004). Additional adjuvants include any compound described inChapter 7 (pp 141-227) of “Vaccine Design, The Subunit and AdjuvantApproach” (eds. Powell, M. F. and Newman, M. J.) PharmaceuticalBiotechnology, Volume 6, Plenum Press (New York). Examples from thiscompendium include Muramyl Dipeptide (MDP) and Montanide 720. Moleculessuch as Poly Inosine:Cytosine (Poly I:C) or plasmid DNA containing CpGmotifs can also be administered as adjuvants in combination withantigens encapsulated in microparticles. In another example, theadjuvant is an agent that facilitates entry of the antigenic compoundinto the cytoplasm of a cell such as listeriolysin, streptolysin or amixture thereof.

Aspects of the present invention provide, in part, an anti-BoNT/Bantibody. As used herein, the term “antibody” means a molecule made inresponse to a particular antigen response and includes, withoutlimitation, polyclonal antibodies, monoclonal antibodies and antigeniccompound-binding fragments of such antibodies, such as, e.g., Fab,F(ab′)₂, Fc, Fd, Fv fragments, and single chain derivatives of the same.Polyclonal antibodies refer to a heterogeneous population of antibodymolecules that contain at least two species of antibody capable ofbinding to a particular antigen. By definition, a polyclonal antibodybinds to at least two different epitopes. Monoclonal antibodies refer toa homogeneous population of antibody molecules that contain only onespecies of antibody capable of binding a particular antigen. Bydefinition, a monoclonal antibody binds to a single epitope. Antibodyalso includes cell-associated antibodies, such as Ig receptors, forexample. In addition, the term “antibody” includes naturally occurringantibodies, as well as non-naturally occurring antibodies, including,for example, chimeric, bi-functional, and humanized antibodies, andrelated synthetic isoforms.

As used herein, the term “anti-BoNT/B antibody” means an anti-BoNT/Bantibody that selectively binds to a BoNT/B. As used herein, the term“selectively” means having a unique effect or influence or reacting inonly one way or with only one thing. As used herein, the term“selectively binds” means the discriminatory binding of the antibody tothe indicated target epitope such that the antibody does notsubstantially cross react with unrelated epitopes. Selective bindingincludes binding properties such as, e.g., binding specificity, bindingaffinity and binding avidity. Binding specificity is the ability of anantibody to discriminate between a molecule containing its epitope and amolecule that does not contain that epitope. An anti-BoNT/B antibodydisclosed in the present specification is characterized by having abinding specificity for its epitope of at least 10-fold greater relativeto a BoNT/B not comprising that epitope. In aspects of this embodiment,an anti-BoNT/B antibody binding specificity for its epitope relative toa BoNT/B not comprising that epitope is, e.g., at least 10-fold greater,at least 100-fold greater, at least 1,000-fold greater or at least10,000-fold greater. Binding affinity is the strength with which anantibody binds its epitope. In an embodiment, an anti-BoNT/B antibodydisclosed in the present specification is characterized by having abinding affinity of at least 1×10⁻⁵ M⁻¹. For example, an anti-BoNT/Bantibody disclosed in the present specification can bind a targetpeptide with a binding affinity of at least 1×10⁻⁵ M⁻¹, at least 1×10⁻⁶M⁻¹, at least 1×10⁻⁷ M⁻¹, at least 1×10⁻⁸ M⁻¹, at least 1×10⁻⁹ M⁻¹, orat least 1×10⁻¹⁰ M⁻¹.

Binding avidity refers to an antibody that can bind more than oneepitope of a target molecule and the binding affinities of theseepitopes. It is envisioned that an anti-BoNT/B antibody disclosed in thepresent specification can selectively bind to any and all epitopes forthat antibody. As used herein, an “epitope” is synonymous with“antigenic determinant” and means the site on a target molecule, suchas, e.g., a peptide, polysaccharide or lipid-containing molecule, thatis bound by a particular antibody or T-cell receptor. The minimal sizeof a peptide epitope, as defined herein, is about five amino acids, anda peptide epitope typically comprises at least eight amino acids. Apeptide epitope may be discontinuous, i.e., it comprises amino acidresidues that are not adjacent in the peptide but are brought togetherinto an epitope by way of the secondary, tertiary, or quaternarystructure of the peptide. Furthermore, it is also noted that an epitopemight comprise a portion of a molecule other than an amino acidsequence, such as, e.g., a carbohydrate moiety, a lipid moiety likelipoproteins or glycolipids, or a chemically-modified amino acid moietylike a phosphorylated amino acid. In aspects of this embodiment, ananti-BoNT/B antibody can selectively bind a BoNT/B epitope comprising atleast five amino acids, at least six amino acids, at least seven aminoacids, at least eight amino acids, at least nine amino acids, at leastten amino acids or at least 20 amino acids. In other aspects of thisembodiment, an anti-BoNT/B antibody can selectively bind a BoNT/Bepitope comprising at most five amino acids, at most six amino acids, atmost seven amino acids, at most eight amino acids, at most nine aminoacids, at most ten amino acids or at most 20 amino acids.

As a non-limiting example, an antibody raised against an antigen willselectively bind a BoNT/B including a portion of, e.g., amino acids610-628 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 1, aminoacids 736-754 of SEQ ID NO: 5, amino acids 778-796 of SEQ ID NO: 1,amino acids 778-796 of SEQ ID NO: 5, amino acids 820-838 of SEQ ID NO:1, amino acids 820-838 of SEQ ID NO: 3, amino acids 820-838 of SEQ IDNO: 5, amino acids 820-838 of SEQ ID NO: 7, amino acids 862-880 of SEQID NO: 1, amino acids 862-880 of SEQ ID NO: 7, amino acids 890-908 ofSEQ ID NO: 1, amino acids 890-908 of SEQ ID NO: 3, amino acids 890-908of SEQ ID NO: 5, amino acids 890-908 of SEQ ID NO: 7, amino acids918-936 of SEQ ID NO: 1, amino acids 918-936 of SEQ ID NO: 3, aminoacids 932-950 of SEQ ID NO: 1, amino acids 932-950 of SEQ ID NO: 5,amino acids 960-978 of SEQ ID NO: 1, amino acids 960-978 of SEQ ID NO:3, amino acids 960-978 of SEQ ID NO: 7, amino acids 974-992 of SEQ IDNO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, aminoacids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,amino acids 1058-1076 of SEQ ID NO: 7, amino acids 1072-1090 of SEQ IDNO: 1, amino acids 1072-1090 of SEQ ID NO: 3, amino acids 1072-1090 ofSEQ ID NO: 7, amino acids 1254-1272 of SEQ ID NO: 1, amino acids1254-1272 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 1, aminoacids 1268-1291 of SEQ ID NO: 3, amino acids 1268-1291 of SEQ ID NO: 5and amino acids 1268-1291 of SEQ ID NO: 7.

As another non-limiting example, an antibody raised against an antigenwill selectively bind a BoNT/B including a portion of, e.g., amino acids616-626 of SEQ ID NO: 1, amino acids 735-745 of SEQ ID NO: 1, aminoacids 735-745 of SEQ ID NO: 5, amino acids 778-789 of SEQ ID NO: 1,amino acids 867-877 of SEQ ID NO: 1, amino acids 867-877 of SEQ ID NO:7, amino acids 895-905 of SEQ ID NO: 1, amino acids 895-905 of SEQ IDNO: 3, amino acids 929-939 of SEQ ID NO: 1, amino acids 974-984 of SEQID NO: 1, amino acids 974-984 of SEQ ID NO: 3, amino acids 974-984 ofSEQ ID NO: 7, amino acids 1039-1049 of SEQ ID NO: 1, amino acids1039-1049 of SEQ ID NO: 5, amino acids 1039-1049 of SEQ ID NO: 7, aminoacids 1065-1075 of SEQ ID NO: 1, amino acids 1065-1075 of SEQ ID NO: 3,amino acids 1065-1075 of SEQ ID NO: 5, amino acids 1065-1075 of SEQ IDNO: 7, amino acids 1269-1281 of SEQ ID NO: 1, amino acids 1269-1281 ofSEQ ID NO: 3, amino acids 1269-1281 of SEQ ID NO: 5 or amino acids1269-1281 of SEQ ID NO: 7.

An anti-BoNT/B antibody disclosed in the present specification can beproduced by a wide variety of methods that are well known in the art.Specific protocols for making and using antibodies as well as detecting,and measuring antibody binding specificity, binding affinity and bindingavidity are known in the art, see, e.g., Harlow & Lane, supra, 1998a;Harlow & Lane, supra, 1998b; Molecular Cloning, A Laboratory Manual,supra, 2001; and Current Protocols in Molecular Biology, supra, 2004;David Anderson et al., Therapeutic Polypeptides, Nucleic Acids EncodingSame, and Methods of Use, U.S. Pat. No. 7,034,132 (Apr. 25, 2005); andBeatriz M. Carreno et al., Antibodies Against CTLA4, U.S. Pat. No.7,034,121 (Apr. 25, 2006).

As a non-limiting example, anti-BoNT/B polyclonal antibodies can beproduced by injecting an individual, such as, e.g., a rabbit, a goat, amouse or another mammal, with one or more injections of an immuneinducing composition disclosed in the present specification. Theresulting anti-BoNT/B polyclonal antibodies produced can be screenedfrom serum of the immunized individual with a BoNT/B peptide disclosedin the present specification using a radioimmunoassay or enzyme-linkedimmunosorbent assay.

As another non-limiting example, an anti-BoNT/B monoclonal antibody canbe produced using a hybridoma method. In this method, an individual,such as, e.g., a mouse, a hamster, or another appropriate hostindividual, is typically exposed to one or more injections of an immuneinducing composition disclosed in the present specification to elicitlymphocytes that produce or are capable of producing anti-BoNT/Bantibodies that will specifically bind to the BoNT/B antigen.Alternatively, the lymphocytes can be immunized in vitro using asuitable cell culture line. Generally, either peripheral bloodlymphocytes are used, if cells of human origin are desired, or spleencells or lymph node cells are used, if non-human mammalian sources aredesired. The lymphocytes are then fused with an immortalized cell lineusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell, see, e.g., Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103. Immortalized cell lines areusually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The culture medium in which the hybridoma cells aregrown can then be assayed for the presence of anti-BoNT/B monoclonalantibodies directed against the BoNT/B antigen disclosed in the presentspecification, see, e.g., Harlow & Lane, supra, 1998a; and Harlow &Lane, supra, 1998b. For example, hybridoma supernatants can be screenedusing anti-BoNT/B-positive sera in an immunoprecipitation assay or by anin vitro binding assay, such as, e.g., a radioimmunoassay (RIA) or anenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). After the desired hybridomacells are identified, the clones can be subcloned by limiting dilutionprocedures until isolate cell line is produced.

As an alternative to preparing monoclonal antibody-secreting hybridomas,an anti-BoNT/B monoclonal antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library, such as,e.g., an antibody phage display library, with a BoNT/B peptide andisolate immunoglobulin library members that bind a BoNT/B peptide. Kitsfor generating and screening phage display libraries are commerciallyavailable, such as, e.g., the Recombinant Phage Antibody System(Pharmacia); and the SurfZAP™ Phage Display Kit (Stratagene).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; McCafferty et al. U.S. Pat. No. 6,172,197;Johnson et al. U.S. Pat. No. 6,140,471; Jespers et al. U.S. Pat. No.6,017,732; Griffiths et al. U.S. Pat. No. 6,010,884; McCafferty et al.U.S. Pat. No. 5,969,108; Griffiths et al. U.S. Pat. No. 5,962,255;Griffiths et al. U.S. Pat. No. 5,885,793; Borrebaeck et al. U.S. Pat.No. 6,027,930; Borrebaeck et al. U.S. Pat. No. 5,712,089.

Non-naturally occurring anti-BoNT/B antibodies can be constructed usingsolid phase peptide synthesis, produced recombinantly or obtained, e.g.,by screening combinatorial libraries consisting of variable heavy chainsand variable light chains as described in, e.g., Huse et al., 246Science 1275-1281 (1989). These and other methods of making, forexample, chimeric, humanized, CDR-grafted, single chain, andbi-functional antibodies are well known to those skilled in the art,see, e.g., Winter and Harris, 14 Immunol. Today 243-246 (1993); Ward etal., 341 Nature 544-546 (1989); Harlow and Lane, supra, 1988a; Hilyardet al., Protein Engineering: A Practical Approach (IRL Press 1992); andBorrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995).

Aspects of the present invention provide, in part, collecting a samplecontaining the anti-BoNT/B antibody or anti-BoNT/B antibody-producingcell. As used herein, the term “sample containing the anti-BoNT/Bantibody or anti-BoNT/B antibody-producing cell” means any biologicalmatter that contains or potentially contains at least one anti-BoNT/Bantibody. It is envisioned that any and all samples that can contain ananti-BoNT/B antibody can be used in this method, including, withoutlimitation, blood, plasma, serum and lymph fluid. It is also envisionedthat any cell capable of producing an anti-BoNT/B antibody can be usedin this method, including, without limitation, a CD8 cells, a CTL cell,a helper T-cell and a B-cell. A variety of well known methods can beused for collecting from an individual a sample containing theanti-BoNT/B antibody or anti-BoNT/B antibody-producing cell, see, e.g.,Harlow & Lane, supra, 1998a; and Harlow & Lane, supra, 1998b. Similarly,a variety of well known methods can be used for processing a sample toisolate an anti-BoNT/B antibody. A procedure for collecting a sample canbe selected based on the type of antibody to be isolated. As anon-limiting example, when isolating anti-BoNT/B polyclonal antibodies,an appropriate sample can be a blood sample containing anti-BoNT/Bantibodies, whereas when isolating monoclonal anti-BoNT/B antibodies, anappropriate sample can be an anti-BoNT/B antibody-producing cell such asa spleen cell.

Aspects of the present invention provide, in part, isolating theanti-BoNT/B antibody from the sample. Methods of isolating ananti-BoNT/B antibody, such as, e.g., anti-BoNT/B polyclonal antibodiesor a anti-BoNT/B monoclonal antibody are well known to those skilled inthe art, see, e.g., Harlow and Lane, supra, 1998a; and Harlow and Lane,supra, 1998b. For example, BoNT/B polyclonal antibodies can be isolatedfrom the sample by well known techniques, such as, e.g., affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific BoNT/B polyclonal antibody sought may be immobilized on acolumn to purify the immune specific antibody by immunoaffinitychromatography. An anti-BoNT/B monoclonal antibody can be isolated fromthe culture medium or ascites fluid by conventional immunoglobulinpurification procedures such as, e.g., protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

Thus, in an embodiment, a method of producing an anti-BoNT/B antibodycomprises the steps of administering to an animal a BoNT/B antigen,collecting from the animal a sample containing the anti-BoNT/B antibodyor anti-BoNT/B antibody-producing cell, and isolating the anti-BoNT/Bantibody from the sample. In an aspect of this embodiment, theanti-BoNT/B antibody is a polyclonal anti-BoNT/B antibody. In anotheraspect of this embodiment, the anti-BoNT/B antibody is a monoclonalanti-BoNT/B antibody.

Aspects of the present invention can also be described as follows:

-   1. A BoNT/B peptide having a length of at most 60 amino acids, the    BoNT/B peptide comprising an amino acid sequence of amino acids    736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino    acids 764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3,    amino acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID    NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of    SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids    848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino    acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7,    amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ    ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids    1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1,    amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ    ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, a conservative    variant of each amino acid sequence thereof, or an immunogenic    fragment of each amino acid sequence thereof.-   2. The BoNT/B peptide according to any one of claim 1, wherein said    BoNT/B peptide has a length of at most 45 amino acids.-   3. The BoNT/B peptide according to any one of claim 1, wherein said    BoNT/B peptide has a length of at most 30 amino acids.-   4. The BoNT/B peptide according to claim 1, wherein the BoNT/B    peptide consists of amino acid sequence of amino acids 736-754 of    SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids    764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino    acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7,    amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID    NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of    SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids    974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino    acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO:    3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of    SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids    1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,    amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of    each amino acid sequence thereof, or an immunogenic fragment of each    amino acid sequence thereof.-   5. A tolerogizing composition comprising a tolerogizing agent    operably linked to a BoNT/B peptide of SEQ ID NO: 1 having a length    of at least five amino acids and at most 60 amino acids, the BoNT/B    peptide comprising an amino acid sequence of amino acids 736-754 of    SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids    764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino    acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7,    amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID    NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of    SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids    974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino    acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO:    3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of    SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids    1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, or    amino acids 1058-1076 of SEQ ID NO: 7.-   6. The tolerogizing composition according to 5, wherein said BoNT/B    peptide has a length of at most 45 amino acids.-   7. The tolerogizing composition according to 5, wherein said BoNT/B    peptide has a length of at most 30 amino acids.-   8. The tolerogizing composition according to 5, wherein the BoNT/B    peptide consists of amino acids 736-754 of SEQ ID NO: 1, amino acids    736-754 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 1, amino    acids 764-782 of SEQ ID NO: 3, amino acids 764-782 of SEQ ID NO: 5,    amino acids 764-782 of SEQ ID NO: 7, amino acids 848-866 of SEQ ID    NO: 1, amino acids 848-866 of SEQ ID NO: 3, amino acids 848-866 of    SEQ ID NO: 5, amino acids 848-866 of SEQ ID NO: 7, amino acids    974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino    acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO:    1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of    SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids    1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3,    amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ    ID NO: 7, a conservative variant of each amino acid sequence    thereof, or an immunogenic fragment of each amino acid sequence    thereof.-   9. The tolerogizing composition according to 5, wherein a portion of    the BoNT/B peptide producing a decrease in an immunological response    comprises at least six consecutive amino acids of 736-754 of SEQ ID    NO: 1, at least six consecutive amino acids of amino acids 736-754    of SEQ ID NO: 5, at least six consecutive amino acids of amino acids    764-782 of SEQ ID NO: 1, at least six consecutive amino acids of    amino acids 764-782 of SEQ ID NO: 3, at least six consecutive amino    acids of amino acids 764-782 of SEQ ID NO: 5, at least six    consecutive amino acids of amino acids 764-782 of SEQ ID NO: 7, at    least six consecutive amino acids of amino acids 848-866 of SEQ ID    NO: 1, at least six consecutive amino acids of amino acids 848-866    of SEQ ID NO: 3, at least six consecutive amino acids of amino acids    848-866 of SEQ ID NO: 5, at least six consecutive amino acids of    amino acids 848-866 of SEQ ID NO: 7, at least six consecutive amino    acids of amino acids 974-992 of SEQ ID NO: 1, at least six    consecutive amino acids of amino acids 974-992 of SEQ ID NO: 3, at    least six consecutive amino acids of amino acids 974-992 of SEQ ID    NO: 7, at least six consecutive amino acids of amino acids 1030-1048    of SEQ ID NO: 1, at least six consecutive amino acids of amino acids    1030-1048 of SEQ ID NO: 3, at least six consecutive amino acids of    amino acids 1030-1048 of SEQ ID NO: 5, at least six consecutive    amino acids of amino acids 1030-1048 of SEQ ID NO: 7, at least six    consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 1, at    least six consecutive amino acids of amino acids 1058-1076 of SEQ ID    NO: 3, at least six consecutive amino acids of amino acids 1058-1076    of SEQ ID NO: 5, at least six consecutive amino acids of amino acids    1058-1076 of SEQ ID NO: 7.-   10. The tolerogizing composition of 5, wherein the tolerogizing    agent is selected from the group consisting of polyethylene glycol    (PEG), monomethoxypolyethylene glycol (mPEG) and polyvinyl alcohol    (PVA).-   11. An immune response inducing composition comprising a BoNT/B    antigen, the BoNT/B antigen comprising a BoNT/B peptide of SEQ ID    NO: 1 having a length of at least five amino acids and at most 60    amino acids, the BoNT/B peptide comprising an amino acid sequence of    amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID    NO: 5, amino acids 764-782 of SEQ ID NO: 1, amino acids 764-782 of    SEQ ID NO: 3, amino acids 764-782 of SEQ ID NO: 5, amino acids    764-782 of SEQ ID NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino    acids 848-866 of SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5,    amino acids 848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID    NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of    SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids    1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5,    amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ    ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids    1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, a    conservative variant of each amino acid sequence thereof, or an    immunogenic fragment of each amino acid sequence thereof.-   12. The immune response inducing composition according to 11,    further comprising a carrier.-   13. The immune response inducing composition according to 12,    wherein the carrier is selected from the group consisting of a    keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin    (THY), a bovine serum albumin (BSA) and a soybean trypsin inhibitor    (STI).-   14. The immune response inducing composition according to 11,    further comprising an adjuvant.-   15. The immune response inducing composition according to 11,    wherein said BoNT/B peptide has a length of at most 45 amino acids.-   16. The immune response inducing composition according to 11,    wherein said BoNT/B peptide has a length of at most 30 amino acids.-   17. The immune response inducing composition according to 11,    wherein the BoNT/B peptide consists of amino acids 736-754 of SEQ ID    NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of    SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids    764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino    acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3,    amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID    NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of    SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids    1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3,    amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ    ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids    1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,    amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of    each amino acid sequence thereof, or an immunogenic fragment of each    amino acid sequence thereof.-   18. The immune response inducing composition according to 11,    wherein a portion of the BoNT/B peptide immunoreactive with an    anti-BoNT antibody comprises at least six consecutive amino acids of    736-754 of SEQ ID NO: 1, at least six consecutive amino acids of    amino acids 736-754 of SEQ ID NO: 5, at least six consecutive amino    acids of amino acids 764-782 of SEQ ID NO: 1, at least six    consecutive amino acids of amino acids 764-782 of SEQ ID NO: 3, at    least six consecutive amino acids of amino acids 764-782 of SEQ ID    NO: 5, at least six consecutive amino acids of amino acids 764-782    of SEQ ID NO: 7, at least six consecutive amino acids of amino acids    848-866 of SEQ ID NO: 1, at least six consecutive amino acids of    amino acids 848-866 of SEQ ID NO: 3, at least six consecutive amino    acids of amino acids 848-866 of SEQ ID NO: 5, at least six    consecutive amino acids of amino acids 848-866 of SEQ ID NO: 7, at    least six consecutive amino acids of amino acids 974-992 of SEQ ID    NO: 1, at least six consecutive amino acids of amino acids 974-992    of SEQ ID NO: 3, at least six consecutive amino acids of amino acids    974-992 of SEQ ID NO: 7, at least six consecutive amino acids of    amino acids 1030-1048 of SEQ ID NO: 1, at least six consecutive    amino acids of amino acids 1030-1048 of SEQ ID NO: 3, at least six    consecutive amino acids of amino acids 1030-1048 of SEQ ID NO: 5, at    least six consecutive amino acids of amino acids 1030-1048 of SEQ ID    NO: 7, at least six consecutive amino acids of amino acids 1058-1076    of SEQ ID NO: 1, at least six consecutive amino acids of amino acids    1058-1076 of SEQ ID NO: 3, at least six consecutive amino acids of    amino acids 1058-1076 of SEQ ID NO: 5, at least six consecutive    amino acids of amino acids 1058-1076 of SEQ ID NO: 7.-   19. A method of determining immunoresistance to botulinum toxin    therapy in an individual, the method comprising the steps of:    -   a) combining a BoNT/B peptide and a test sample under conditions        suitable for the selective binding of the BoNT/B peptide to an        anti-BoNT antibody, the BoNT/B peptide having a length of at        least 5 amino acids and at most 60 amino acids and comprising        the amino acid sequence of amino acids 736-754 of SEQ ID NO: 1,        amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of SEQ        ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids        764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7,        amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ        ID NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids        848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1,        amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ        ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids        1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO:        5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076        of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino        acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID        NO: 7, a conservative variant of each amino acid sequence        thereof, or an immunogenic fragment of each amino acid sequence        thereof; and    -   b) detecting the presence of an anti-BoNT antibody-BoNT/B        peptide complex, the antibody-peptide complex formed by the        selective binding of an anti-BoNT antibody and the BoNT/B        peptide;    -   where the presence of the anti-BoNT antibody-BoNT/B peptide        complex is indicative of immunoresistance to a BoNT therapy.-   20. The method according to claim 19, wherein the method comprises a    further step of correlating the amount of an antibody-peptide    complex formed from the test sample relative to the amount of an    antibody-peptide complex formed by the BoNT/B peptide combined to a    control sample.-   21. The method according to claim 19, wherein a portion of the    BoNT/B peptide immunoreactive with an anti-BoNT antibody comprises    at least six consecutive amino acids of 736-754 of SEQ ID NO: 1, at    least six consecutive amino acids of amino acids 736-754 of SEQ ID    NO: 5, at least six consecutive amino acids of amino acids 764-782    of SEQ ID NO: 1, at least six consecutive amino acids of amino acids    764-782 of SEQ ID NO: 3, at least six consecutive amino acids of    amino acids 764-782 of SEQ ID NO: 5, at least six consecutive amino    acids of amino acids 764-782 of SEQ ID NO: 7, at least six    consecutive amino acids of amino acids 848-866 of SEQ ID NO: 1, at    least six consecutive amino acids of amino acids 848-866 of SEQ ID    NO: 3, at least six consecutive amino acids of amino acids 848-866    of SEQ ID NO: 5, at least six consecutive amino acids of amino acids    848-866 of SEQ ID NO: 7, at least six consecutive amino acids of    amino acids 974-992 of SEQ ID NO: 1, at least six consecutive amino    acids of amino acids 974-992 of SEQ ID NO: 3, at least six    consecutive amino acids of amino acids 974-992 of SEQ ID NO: 7, at    least six consecutive amino acids of amino acids 1030-1048 of SEQ ID    NO: 1, at least six consecutive amino acids of amino acids 1030-1048    of SEQ ID NO: 3, at least six consecutive amino acids of amino acids    1030-1048 of SEQ ID NO: 5, at least six consecutive amino acids of    amino acids 1030-1048 of SEQ ID NO: 7, at least six consecutive    amino acids of amino acids 1058-1076 of SEQ ID NO: 1, at least six    consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 3, at    least six consecutive amino acids of amino acids 1058-1076 of SEQ ID    NO: 5, at least six consecutive amino acids of amino acids 1058-1076    of SEQ ID NO: 7.-   22. The method according to claim 19, wherein the test sample    comprises blood.-   23. The method according to claim 19, wherein the test sample    comprises serum.-   24. The method according to claim 23, wherein the test sample    comprises an IgG antibody component separated from the serum.-   25. The method according to claim 19, wherein the immunoresistance    to botulinum toxin therapy is selected from the group consisting of    a BoNT/A immunoresistance, a BoNT/B immunoresistance, a BoNT/C1    immunoresistance, a BoNT/D immunoresistance, a BoNT/E    immunoresistance, a BoNT/F immunoresistance and a BoNT/G    immunoresistance.-   26. The method according to claim 25, wherein the immunoresistance    to botulinum toxin therapy is a BoNT/B immunoresistance.-   27. The method according to claim 19, wherein the individual is a    human.-   28. The method according to claim 19, wherein the presence of an    anti-BoNT antibody-BoNT/B peptide complex is determined    qualitatively.-   29. The method according to claim 19, wherein the presence of an    anti-BoNT antibody-BoNT/B peptide complex is determined    quantitatively.-   30. The method according to claim 19, wherein the presence of an    anti-BoNT antibody-BoNT/B peptide complex is determined using a    competitive assay.-   31. The method according to claim 19, wherein the presence of an    anti-BoNT antibody-BoNT/B peptide complex is determined using a    non-competitive assay.-   32. The method according to claim 19, wherein the presence of an    anti-BoNT antibody-BoNT/B peptide complex is determined by an assay    format selected from the group consisting of a radioimmunoassay, an    enzyme-linked immunosorbent assay, an enzyme immunoassay, a    fluorescence immunoassay, and a luminescent immunoassay.-   33. The method according to claim 19, wherein the presence of at    least 10% complex formation of an anti-BoNT antibody to a BoNT/B    peptide is indicative of a BoNT immunoresistance.-   34. The method according to claim 19, wherein the presence of at    least 30% complex formation of an anti-BoNT antibody to a BoNT/B    peptide is indicative of a BoNT immunoresistance.-   35. The method according to claim 19, wherein the presence of at    least 50% complex formation of an anti-BoNT antibody to a BoNT/B    peptide is indicative of a BoNT immunoresistance.-   36. The method according to claim 19, wherein the presence of at    most 10% complex formation of an anti-BoNT antibody to a BoNT/B    peptide is indicative of a BoNT immunoresistance.-   37. The method according to claim 19, wherein the presence of at    most 30% complex formation of an anti-BoNT antibody to a BoNT/B    peptide is indicative of a BoNT immunoresistance.-   38. The method according to claim 19, wherein the presence of at    most 50% complex formation of an anti-BoNT antibody to a BoNT/B    peptide is indicative of a BoNT immunoresistance.-   39. An anti-BoNT immunoapheresis method of treating immunoresistance    to a botulinum toxin therapy in an individual, the method comprising    the steps of:    -   a) contacting an anti-BoNT antibody containing component from        the individual extracorporeally with a BoNT/B peptide        immunosorbent under conditions suitable for the selective        binding of the BoNT/B peptide to the anti-BoNT antibody, the        BoNT/B peptide having a length of at least 5 amino acids and at        most 60 amino acids; and    -   b) returning the anti-BoNT antibody depleted component back into        the individual.-   40. The immunoapheresis method according to claim 39, wherein the    anti-BoNT antibody containing component comprises blood.-   41. The immunoapheresis method according to claim 39, wherein the    anti-BoNT antibody containing component comprises serum.-   42. The immunoapheresis method according to claim 41, wherein an IgG    antibody component from the serum is contacted a BoNT/B peptide    immunosorbent.-   43. The immunoapheresis method according to claim 39, wherein the    immunoresistance to botulinum toxin therapy is selected from the    group consisting of a BoNT/A immunoresistance, a BoNT/B    immunoresistance, a BoNT/C1 immunoresistance, a BoNT/D    immunoresistance, a BoNT/E immunoresistance, a BoNT/F    immunoresistance and a BoNT/G immunoresistance.-   44. The method according to claim 43, wherein the immunoresistance    to botulinum toxin therapy is a BoNT/B immunoresistance.-   45. The immunoapheresis method according to claim 39, wherein the    individual is a human.-   46. The immunoapheresis method according to claim 39, wherein the    amount of anti-BoNT antibodies removed from an anti-BoNT antibody    containing component is at least 10% of the anti-BoNT antibodies    from the anti-BoNT antibody containing component.-   47. The immunoapheresis method according to claim 39, wherein the    amount of anti-BoNT antibodies removed from an anti-BoNT antibody    containing component is at least 30% of the anti-BoNT antibodies    from the anti-BoNT antibody containing component.-   48. The immunoapheresis method according to claim 39, wherein the    amount of anti-BoNT antibodies removed from an anti-BoNT antibody    containing component is at least 50% of the anti-BoNT antibodies    from the anti-BoNT antibody containing component.-   49. The immunoapheresis method according to claim 39, wherein the    amount of anti-BoNT antibodies removed from an anti-BoNT antibody    containing component is at most 10% of the anti-BoNT antibodies from    the anti-BoNT antibody containing component.-   50. The immunoapheresis method according to claim 39, wherein the    amount of anti-BoNT antibodies removed from an anti-BoNT antibody    containing component is at most 30% of the anti-BoNT antibodies from    the anti-BoNT antibody containing component.-   51. The immunoapheresis method according to claim 39, wherein the    amount of anti-BoNT antibodies removed from an anti-BoNT antibody    containing component is at most 50% of the anti-BoNT antibodies from    the anti-BoNT antibody containing component.-   52. A method of treating immunoresistance to a botulinum toxin    therapy in an individual, the method comprising the step of    administering to the individual a tolerogizing composition, the    tolerogizing composition comprising a tolerogizing agent conjugated    to a BoNT/B peptide the BoNT/B peptide having a length of at least 5    amino acids and at most 60 amino acids, where the administration of    the tolerogizing composition producing a decrease in an    immunological response against the botulinum toxin therapy in the    individual.-   53. The method according to 52, wherein the immunoresistance being    treated is selected from the group consisting of a BoNT/A    immunoresistance, a BoNT/B immunoresistance, a BoNT/C1    immunoresistance, a BoNT/D immunoresistance, a BoNT/E    immunoresistance, a BoNT/F immunoresistance and a BoNT/G    immunoresistance.-   54. The method according to 53, wherein the immunoresistance being    treated is a BoNT/B immunoresistance.-   55. The method according to 54, wherein the individual is a human.-   56. The method according to 52, wherein the BoNT/B peptide    comprising an amino acid sequence of amino acids amino acids 736-754    of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids    764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino    acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7,    amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID    NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of    SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids    974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino    acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO:    3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of    SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids    1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,    amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of    each amino acid sequence thereof, or an immunogenic fragment of each    amino acid sequence thereof.-   57. The method according to 56, wherein the amino acid sequence is    selected from the group consisting of amino acids 736-754 of SEQ ID    NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of    SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids    764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino    acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3,    amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID    NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of    SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids    1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3,    amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ    ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids    1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,    amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of    each amino acid sequence thereof, or an immunogenic fragment of each    amino acid sequence thereof.-   58. The method according to 56, wherein a portion of the BoNT/B    peptide producing the decreased immunological response comprises at    least six consecutive amino acids of 736-754 of SEQ ID NO: 1, at    least six consecutive amino acids of amino acids 736-754 of SEQ ID    NO: 5, at least six consecutive amino acids of amino acids 764-782    of SEQ ID NO: 1, at least six consecutive amino acids of amino acids    764-782 of SEQ ID NO: 3, at least six consecutive amino acids of    amino acids 764-782 of SEQ ID NO: 5, at least six consecutive amino    acids of amino acids 764-782 of SEQ ID NO: 7, at least six    consecutive amino acids of amino acids 848-866 of SEQ ID NO: 1, at    least six consecutive amino acids of amino acids 848-866 of SEQ ID    NO: 3, at least six consecutive amino acids of amino acids 848-866    of SEQ ID NO: 5, at least six consecutive amino acids of amino acids    848-866 of SEQ ID NO: 7, at least six consecutive amino acids of    amino acids 974-992 of SEQ ID NO: 1, at least six consecutive amino    acids of amino acids 974-992 of SEQ ID NO: 3, at least six    consecutive amino acids of amino acids 974-992 of SEQ ID NO: 7, at    least six consecutive amino acids of amino acids 1030-1048 of SEQ ID    NO: 1, at least six consecutive amino acids of amino acids 1030-1048    of SEQ ID NO: 3, at least six consecutive amino acids of amino acids    1030-1048 of SEQ ID NO: 5, at least six consecutive amino acids of    amino acids 1030-1048 of SEQ ID NO: 7, at least six consecutive    amino acids of amino acids 1058-1076 of SEQ ID NO: 1, at least six    consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 3, at    least six consecutive amino acids of amino acids 1058-1076 of SEQ ID    NO: 5, at least six consecutive amino acids of amino acids 1058-1076    of SEQ ID NO: 7.-   59. The method according to 52, wherein the tolerogizing composition    is administered to the individual systemically.-   60. The method according to 52, wherein the tolerogizing composition    is administered to the individual peripherally.-   61. The method according to 52, wherein the tolerogizing composition    is administered to the individual prior to administering a botulinum    toxin therapy.-   62. The method according to 52, wherein the tolerogizing composition    is administered to the individual during a course of botulinum toxin    therapy.-   63. The method according to 52, wherein the tolerogizing composition    is administered to the individual after an onset of a BoNT    immunoresistance.-   64. The method according to 52, wherein the route of administration    is selected from the group consisting of an oral administration, an    injection and an implant.-   65. A method of inducing a BoNT/B immune response in an individual,    the method comprising the step of administering to the individual an    immune response inducing composition comprising an adjuvant and a    BoNT/B antigen, where administration of the immune response inducing    composition produces an immune response in the individual;    -   wherein the BoNT/B antigen comprises a BoNT/B peptide having a        length of at least 5 amino acids and at most 60 amino acids and        comprises the amino acid sequence of amino acids 736-754 of SEQ        ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids        764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3,        amino acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ        ID NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino acids        848-866 of SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5,        amino acids 848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ        ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids        974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1,        amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of        SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids        1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO:        3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076        of SEQ ID NO: 7, a conservative variant of each amino acid        sequence thereof, or an immunogenic fragment of each amino acid        sequence thereof.-   66. The method according to 65, wherein the amino acid sequence is    selected from the group consisting of amino acids 736-754 of SEQ ID    NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of    SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids    764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino    acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3,    amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID    NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of    SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids    1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3,    amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ    ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids    1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5,    amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of    each amino acid sequence thereof, or an immunogenic fragment of each    amino acid sequence thereof.-   67. The method according to 65, wherein a portion of the BoNT/B    peptide producing the immune response comprises at least six    consecutive amino acids of 736-754 of SEQ ID NO: 1, at least six    consecutive amino acids of amino acids 736-754 of SEQ ID NO: 5, at    least six consecutive amino acids of amino acids 764-782 of SEQ ID    NO: 1, at least six consecutive amino acids of amino acids 764-782    of SEQ ID NO: 3, at least six consecutive amino acids of amino acids    764-782 of SEQ ID NO: 5, at least six consecutive amino acids of    amino acids 764-782 of SEQ ID NO: 7, at least six consecutive amino    acids of amino acids 848-866 of SEQ ID NO: 1, at least six    consecutive amino acids of amino acids 848-866 of SEQ ID NO: 3, at    least six consecutive amino acids of amino acids 848-866 of SEQ ID    NO: 5, at least six consecutive amino acids of amino acids 848-866    of SEQ ID NO: 7, at least six consecutive amino acids of amino acids    974-992 of SEQ ID NO: 1, at least six consecutive amino acids of    amino acids 974-992 of SEQ ID NO: 3, at least six consecutive amino    acids of amino acids 974-992 of SEQ ID NO: 7, at least six    consecutive amino acids of amino acids 1030-1048 of SEQ ID NO: 1, at    least six consecutive amino acids of amino acids 1030-1048 of SEQ ID    NO: 3, at least six consecutive amino acids of amino acids 1030-1048    of SEQ ID NO: 5, at least six consecutive amino acids of amino acids    1030-1048 of SEQ ID NO: 7, at least six consecutive amino acids of    amino acids 1058-1076 of SEQ ID NO: 1, at least six consecutive    amino acids of amino acids 1058-1076 of SEQ ID NO: 3, at least six    consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 5, at    least six consecutive amino acids of amino acids 1058-1076 of SEQ ID    NO: 7.-   68. The method according to 65, wherein the method further comprises    collecting from the individual a sample containing an anti-BoNT/B    antibody or anti-BoNT/B antibody-producing cell; and isolating the    anti-BoNT/B antibody from the sample.-   69. The method according to 68, wherein anti-BoNT/B antibody    isolated is a polyclonal anti-BoNT/B antibody.-   70. The method according to 68, wherein anti-BoNT/B antibody    isolated is a monoclonal anti-BoNT/B antibody.-   71. The method according to 65, wherein the BoNT/B antigen comprises    a BoNT/B peptide having a length of at least 5 amino acids and at    most 60 amino acids operably linked to a carrier.-   72. The method according to 71, wherein the. carrier is selected    from the group consisting of a keyhole limpet hemacyanin (KLH), an    ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA)    and a soybean trypsin inhibitor (STI).-   73. The method according to 65, wherein the. adjuvant is selected    from the group consisting of a Freund's complete adjuvant (FCA), a    Freund's incomplete adjuvant (FIA), a sapogenin glycoside, a    carbopol a N-acetylmuramyl-L-alanyl-D-isoglutamine and a    lipopolysaccharide.

EXAMPLES Example 1 Synthesis of BoNT/B Peptides

BoNT/B peptides were prepared by solid-phase peptide synthesis on abenzyloxybenzyl alcohol resin to which 9-fluorenylmethylcar-bonyl(Fmoc)-glycine had been coupled. The N^(α)-Fmoc amino acid derivativeswere obtained from Vega or from Peninsula Laboratories. The side-chainprotecting groups were as follows: for aspartic acid, β-tert-butylester; for glutamic acid, γ-tert-butyl ester; for cysteine,S-tert-butyl; for histidine, im-trityl; for lysine,ε-tert-butoxycarbonyl; for serine, threonine, or tyrosine, O-tert-butyl;for arginine, N^(ω)-methoxy-2,3,6-trimethylphenyl-sulfonyl. Removal ofthe N^(α)-Fmoc group before each coupling was done by treatment of thepeptide resin with 20% piperidine in dimethylformamide (DMF) for 10 min.This was followed by washing (3 times each, 30 sec) with DMF, methanol,and then DMF. Coupling of consecutive amino acids was done for 2 hr byusing 3-molar excess of each of the Fmoc amino acid derivatives,diisopropylcarbodiimide in DMF/CH₂Cl₂, 1:1 (vol/vol) and1-hydroxybenzotriazole. The resin was then washed with DMF and methanol(three times each, 30 sec), followed by two 30-sec washes of CH₂Cl₂. Thecompletion of coupling after each residue was monitored by ninhydrin,and recoupling was repeated when necessary. After the last cycle anddeprotection of the Fmoc-group, the peptide was cleaved from the resinby treatment (2.5 hr) with 55% trifluoroacetic acid in CH₂Cl₂, and thesolvent was removed on a rotary evaporator. The peptide was washed threetimes with cold ether, dissolved in water, and freeze-dried. Theproducts were purified by chromatography on CM-Sephadex C50 orDEAE-Sephadex A50. The peptides thus obtained were homogeneous byhigh-voltage paper electrophoresis and by analytical HPLC on a C18column using a gradient of 0.1% trifluoracetic acid in water/0.1%trifluoracetic acid in acetonitrile. The amino acid sequence for eachpeptide was determined and found to have an amino acid compositionconsistent with that expected from BoNT/B.

The peptides had a uniform size of 19 residues each (except for peptideC31 which was 24 residues) and overlapped consecutively by 5 residues.Table 2 shows the synthetic consecutive overlapping peptides of theH_(N) domain of BoNT/B having the indicated residues of SEQ ID NO: 1.Regions of overlap with adjacent peptides are underlined and bolded.Table 3 shows synthetic consecutive overlapping peptides of the H_(C)domain of BoNT/B having the indicated residues of SEQ ID NO: 1. Theregions of overlap with adjacent BoNT/B peptides are underlined andbolded.

TABLE 2 Synthetic Overlapping Peptides from H_(N) Domain of the BoNT/BHeavy Chain Pep- tide Num- Sequence ber position Amino acid sequence N1442-460 A P G I C I D V D N E D L F F I A D K N2 456-474 F I A D K  N SF S D D L S K N E R I E N3 470-488 N E R I E  Y N T Q S N Y I EN D F P I N4 484-502 N D F P I  N E L I L D T D L I S K I E N5 498-516I S K I E  L P S E N T E S L T D F N V N6 512-530 T D F N V  D V P V Y EK Q P A I K K I N7 526-544 A I K K I  F T D E N T I F Q Y L Y S Q N8540-558 Y L Y S Q  T F P L D I R D I S L T S S N9 554-572 S L T S S  F DD A L L F S N K V Y S F N10 568-586 K V Y S F  F S M D Y I K T AN K V V E N11 582-600 N K V V E  A G L F A G W V K Q I V N D N12 596-614Q I V N D  F V I E A N K S N T M D K I N13 610-628 T M D K I  A D I S LI V P Y I G L A L N14 624-642 I G L A L  N V G N E T A K G N F E N A N15638-656 N F E N A  F E I A G A S I L L E F I P N16 652-670 L E F I P  EL L I P V V G A F L L E S N17 666-684 F L L E S  Y I D N K N K I IK T I D N N18 680-698 K T I D N  A L T K R N E K W S D M Y G N19 694-712S D M Y G  L I V A Q W L S T V N T Q F N20 708-726 V N T Q F  Y T I K EG M Y K A L N Y Q N21 722-740 A L N Y Q  A Q A L E E I I K Y R Y N I N22736-754 Y R Y N I  Y S E K E K S N I N I D F N N23 750-768 N I D F N  DI N S K L N E G I N Q A I N24 764-782 I N Q A I  D N I N N F I N GC S V S Y N25 778-796 C S V S Y  L M K K M I P L A V E K L L N26 792-810V E K L L  D F D N T L K K N L L N Y I N27 806-824 L L N Y I  D E N K LY L I G S A E Y E N28 820-838 S A E Y E  K S K V N K Y L K T I M P F N29834-852 T I M P F  D L S I Y T N D T I L I E M

TABLE 3 Synthetic Overlapping Peptides from H_(C) Domain of the BoNT/BHeavy Chain Pep- tide Num- Peptide ber Number Peptide Number C1 848-866I L I E M  F N K Y N S E I L N N I I L C2 862-880 N N I I L  N L R Y K DN N L I D L S G C3 876-894 I D L S G  Y G A K V E V Y D G V E L N C4890-908 G V E L N  D K N Q F K L T S S A N S K C5 904-922 S A N S K  I RV T Q N Q N I I F N S V C6 918-936 I F N S V  F L D F S V S F WI R I P K C7 932-950 I R I P K  Y K N D G I Q N Y I H N E Y C8 946-964I H N E Y  T I I N C M K N N S G W K I C9 960-978 S G W K I  S I R G N RI I W T L I D I C10 974-992 T L I D I  N G K T K S V F F E Y N I R C11 988- E Y N I R  E D I S E Y I N R W F F V T 1006 C12 1002- W F F V T  IT N N L N N A K I Y I N G 1020 C13 1016- I Y I N G  K L E S N T D I KD I R E V 1034 C14 1030- D I R E V  I A N G E I I F K L D G D I 1048 C151044- L D G D I  D R T Q F I W M K Y F S I F 1062 C16 1058- Y F S I F  NT E L S Q S N I E E R Y K 1076 C17 1072- E E R Y K  I Q S Y S E Y L KD F W G N 1090 C18 1086- D F W G N  P L M Y N K E Y Y M F N A G 1104 C191100- M F N A G  N K N S Y I K L K K D S P V 1118 C20 1114- K D S P V  GE I L T R S K Y N Q N S K 1132 C21 1128- N Q N S K  Y I N Y R D L Y IG E K F I 1146 C22 1142- G E K F I  I R R K S N S Q S I N D D I 1160 C231156- I N D D I  V R K E D Y I Y L D F F N L 1174 C24 1170- D F F N L  NQ E W R V Y T Y K Y F K K 1188 C25 1184- K Y F K K  E E E K L F L A PI S D S D 1202 C26 1198- I S D S D  E F Y N T I Q I K E Y D E Q 1216 C271212- E Y D E Q  P T Y S C Q L L F K K D E E 1230 C28 1226- K K D E E  ST D E I G L I G I H R F Y 1244 C29 1240- I H R F Y  E S G I V F E E YK D Y F C 1258 C30 1254- K D Y F C  I S K W Y L K E V K R K P Y 1272 C311268- K R K P Y  N L K L G C N W Q F I P K D 1291 E G W T E

Example 2 Mapping of Human Anti-Pentavalent Botulinum Toxoid AntibodiesUsing Synthetic BoNT/B Peptides

This example shows antigenic mapping of BoNT/B with humananti-pentavalent botulinum toxoid antisera using the using 60 syntheticBoNT/B peptides that encompass the H_(N) and H_(C) domains of BoNT/B.

Human antisera against BoNT/B were prepared by immunizing humanvolunteers with a toxoid preparation made from pentavalent toxoid(BoNT/A, /B, /C1, /D and /E), see, e.g., Zouhair M. Atassi et al.Mapping of the antibody-binding regions on botulinum neurotoxin H-chaindomain 855-1296 with anti-toxin antibodies from three host species,15(7) J. Prot. Chem. 691-700 (1996). The BoNT/B peptide binding assaydescribed below was performed using IgG fractions of these antisera. Foruse as a control, an IgG fraction was prepared using pre-immune humanserum. BoNT/B was purchased from (Metabiologics, Madison, Wis.).

To conduct a BoNT/B peptide binding assay, BoNT/B peptides (2.5 μg in 50μl of PBS) or active BoNT/B (1 μg in 50 μl of PBS) were added to thewells of flexible polyvinyl chloride 96-well plates (Becton Dickinson,San Jose, Calif.) and allowed to bind for 18 hours at 4° C. Afterwashing five times with PBS, the plates were blocked for 1 hour at 37°C. with 1% bovine serum albumin (BSA) in PBS. Aliquots (50 μl) ofanti-toxin antisera that had been prediluted with 0.1% BSA in PBS(dilutions were human IgG fraction, 1:1000 and 1:2000 (vol/vol)) werepipetted into the appropriate wells and allowed to react for 3 hours at37° C. The wells were washed five times with PBS before adding 50 μl ofaffinity-purified rabbit Ig fraction against human IgG and IgM (DakoCorporation; Carpinteria, CA) diluted 1:1000 with 0.1% BSA in PBS to thewells of the plate, and incubating for 2 hours at 37° C.

The wells were then washed five times with PBS, and 50 μl of¹²⁵I-labeled Protein A (2×10⁵ cpm in 0.1% BSA in PBS) was distributed tothe wells and allowed to incubate for 2 hours at room temperature.Finally, the plates were washed thoroughly to remove unboundradioactivity, the individual wells were cut out and transferred intoseparate tubes, and bound radioactivity was counted in a gamma-counter(1277 Gamma Master, LKB, Finland). Controls included binding ofpreimmune sera to BoNT/B and its peptides, as well as binding of immunesera to BSA and BoNT/B-unrelated peptides. Assays were performed intriplicate. Results of the triplicate analyses were expressed as mean ofnet cpm±SD, after correction for nonspecific binding in control wellsthat were coated with BSA and unrelated peptides.

Human anti-BoNT antisera raised against the pentavalent toxoid wasobserved to bind to several BoNT/B peptides (FIG. 1). Very high levelsof antibodies were bound at both dilutions by peptide C10. Antibodieswere also bound by the following peptides in decreasing order: C4, N22,C16, C9, C31, N13, C2, C14, N25 and C6/C7 overlap. The remaining BoNT/Bpeptides bound little or no antibodies. Nonimmune human IgG did not bindto any BoNT/B peptides, and human anti-BoNT antisera showed no antibodybinding to unrelated proteins and peptides. The results define antigenicportions of the H_(N) and H_(C) domains of BoNT/B.

The three-dimensional structure of BoNT/B reveals the solvent-exposedportions of the primary BoNT/B sequence, see, e.g., S. Swaminathan andS. Eswaramoorthy, Structural Analysis of the Catalytic and Binding Sitesof Clostridium botulinum Neurotoxin B, 7 Nat. Struct. Biol. 693-699(2000). Comparison with the results obtained in the present studyrevealed that the immunodominant antibody-binding regions reside onsurface locations on the heavy chain of BoNT/B. In sum, these resultsdemonstrate that BoNT/B peptides N13, N22, N25, C2, C4, C6/C7 overlap,C9, C10, C14, C16 and C31 were recognized by human anti-pentavalentbotulinum toxoid antisera.

Example 3 Mapping of Horse Anti-BoNT/B Antibodies Using Synthetic BoNT/BPeptides

This example describes antigenic mapping of BoNT/B with horseanti-BoNT/B antisera using 60 BoNT/B synthetic peptides that encompassthe H_(N) and H_(C) domains of BoNT/B.

Hyperimmune horse anti-BoNT/B antisera were prepared by subcutaneousimmunization, in multiple sites every two weeks for over a year, with aformaldehyde-inactivated BoNT/B in RIBI adjuvant. The antisera tested inthe binding studies were obtained after four injections according toprocedures described in Atassi et al., supra, 1996. For use as controls,preimmune horse sera were obtained from the animals before immunization.

BoNT/B peptide binding assays were performed as described in Example 2,except that the dilution for horse antisera was 1:500 (vol/vol) and1:1000 (vol/vol). The secondary antibodies were affinity purified rabbitanti-horse IgG (Accurate Chemical & Scientific Corporation, Weston,N.Y.) and were diluted 1:500 (vol/vol) before use.

The binding profile of horse anti-BoNT/B antibodies to the BoNT/Bpeptides is shown in FIG. 2. Antibodies were bound, in decreasingamounts to peptides N28, C7/C8 overlap, N25/N26 overlap, C10, N22, C4,C20, C27 and N20. At least 10 other peptides showed very low binding atthe antiserum concentration 1:500 (vol/vol), which disappeared at thedilution of 1:1000 vol/vol (FIG. 2). The remaining peptides possessedvery low or no binding. Antibodies were also bound by BoNT/B but not toany unrelated (to BoNT/B) proteins or peptides. In sum, these resultsdemonstrate that peptides N20, N22, N25/N26 overlap, N28, C4, C7/C8overlap, C10, C20 and C27 were recognized by horse anti-BoNT/B antisera.

Example 4 Mapping of Mouse Anti-BoNT/B Antibodies Using Synthetic BoNT/BPeptides

This example describes antigenic mapping of BoNT/B with mouseanti-BoNT/B antisera using 60 BoNT/B synthetic peptides that encompassthe H_(N) and H_(C) domains of BoNT/B.

Mouse anti-BoNT antisera were prepared in outbred ICR mice bysubcutaneous immunization with 10 μg formaldehyde-inactivated BoNT/B incomplete Freund's adjuvant and were boosted every four weeks with asimilar dose of inactivated toxin in incomplete Freund's adjuvant.Antisera used in these studies were obtained 158 days after the firstinjection, see, e.g., Atassi et al., supra, 1996. Preimmune mouse serawas employed as a control. Mice were purchased from the National CancerInstitute, and Jackson Laboratory (Bar Harbor, Me.).

BoNT/B peptide binding assays were performed as described in Example 2,except that the dilution for antisera of outbred mice was 1:1000 and1:2000 (vol/vol). The secondary antibodies (mouse IgG (H+L)+IgM (Muchain) were obtained from Accurate Chemical & Scientific Corporation(Westbury, N.Y.) and were diluted 1:2000 (vol/vol).

As shown in FIG. 3, mouse anti-BoNT antisera were observed to bind toseveral BoNT/B peptides. Antibodies were bound in decreasing amounts tothe following peptides: C10, C26, C29, N2/N3 overlap, C16/C17 overlap,N22, C31, C14, C7 and C1/C2 overlap. Very low or no binding wasexhibited by the remaining BoNT/B peptides. The mouse anti-BoNT antiseraexhibited no antibody binding to unrelated proteins and peptides.Preimmune sera from the same mice did not bind to any of the H_(N) orH_(C) peptides. In sum, these results demonstrate that peptides N2/N3overlap, N22, C1/C2 overlap, C7, C10, C14, C16/C17 overlap, C26, C29 andC31 were recognized by mouse anti-BoNT/B antisera.

Example 5 Comparison of BoNT/B Antigenicity between Human Horse andMouse Antisera

This example defines several common immunogenic regions of BoNT/B byantigen mapping obtained with antisera from three different species.

A comparison of the recognition profiles by human, horse and mouseanti-BoNT/B antibodies of the BoNT/B peptides is summarized in Tables 4and 5 and shown graphically in FIG. 4. The results showed that theantisera of the three host species had for the most part similarrecognition profiles with minor shifts to the right or to the left.There were, however, regions that were recognized either by two of thethree antisera species or that were unique to one species. Theantibodies of all three species recognized peptides N22, N25, C7, C10,C16 and C31. The recognition profiles are even more similar if shifts ofbinding regions by one peptide up or down are taken into account (Table2), because such shifts can happen when the boundaries of the sitesshift by 1-2 residues to the left or to the right. Shifts to the rightor to the left are seen in regions N25/N26, C1/C2, C6/C7, C13/C14 andC30/C31. BoNT/B peptides N10/N11 and N17 bound low amounts of antibodiesonly with human sera; peptide C8 by horse antibodies, while peptidesN2/N3, C22, C26 and C29 bound moderate-to-high amounts of mouseantibodies. In sum, this example shows that anti-BoNT antibodies fromhuman, mouse, horse and chicken recognize several common immunogenicregions from the H_(N) and H_(C) domains of the BoNT/B.

TABLE 4 Summary of Antibodies that Recognize BoNT/B H_(N) PeptidesSequence Position anti-BoNT/B Antisera Peptide No. (residues of SEQ IDNO: 1) Human Horse Mouse N1 442-460 − − − N2 456-474 − − − N3 470-488 ±− ++ N4 484-502 − ± +++ N5 498-516 − ± − N6 512-530 − ± − N7 526-544 − ±− N8 540-558 ± − − N9 554-572 − − − N10 568-586 + − − N11 582-600 + − −N12 596-614 − − − N13 610-628 ++ + − N14 624-642 ± − − N15 638-656 + − +N16 652-670 − − − N17 666-684 + − − N18 680-698 − − − N19 694-712 − ± −N20 708-726 ± + − N21 722-740 ± − + N22 736-754 +++ ++ +++ N23 750-768 −− − N24 764-782 − − ± N25 778-796 ++ +++ + N26 792-810 − ++ − N27806-824 − − − N28 820-838 + +++ − N29 834-852 ± ± − Active BoNT/B —+++++ +++++ +++++ (+) or (−) signs are based on net cpm values anddenote the following: (−), less than 1,500 cpm; (Å), 1,500-3,000 cpm;(+), 3,000-7,000 cpm; (++), 7,000-15,000 cpm; (+++), 15,000-25,000 cpm;(++++), 25,000-35,000 cpm; (+++++), exceeding 35,000 cpm. Net cpm valueswere derived from antisera that gave the highest binding.

TABLE 5 Summary of Antibodies that Recognize BoNT/B H_(N) PeptidesSequence Position anti-BoNT/B Antisera Peptide No. (Residues of SEQ IDNO: 1) Human Horse Mouse C1 848-866 − + ++ C2 862-880 ++ ± + C3876-894 + ± − C4 890-908 ++++ ++ − C5 904-922 − − − C6 918-936 ++ − − C7932-950 + +++ ++ C8 946-964 − ++ − C9 960-978 ++ − − C10 974-992 +++++++ +++++ C11  988-1006 − − − C12 1002-1020 − − − C13 1016-1034 − ± − C141030-1048 ++ − ++ C15 1044-1062 − − − C16 1058-1076 +++ + +++ C171072-1090 + ± +++ C18 1086-1104 − − − C19 1100-1118 + + − C20 1114-1132− ++ − C21 1128-1146 − + ++ C22 1142-1160 ± − +++ C23 1156-1174 − − ±C24 1170-1188 + + − C25 1184-1202 − − − C26 1198-1216 − ± ++++ C271212-1230 − − − C28 1226-1244 − − − C29 1240-1258 − − +++ C30 1254-1272++ + − C31 1268-1291 ++ ± +++ Active — +++++ +++++ +++++ BoNT/B (+) or(−) signs are based on net cpm values and denote the following: (−),less than 1,500 cpm; (Å), 1,500-3,000 cpm; (+), 3,000-7,000 cpm; (++),7,000-15,000 cpm; (+++), 15,000-25,000 cpm; (++++), 25,000-35,000 cpm;(+++++), exceeding 35,000 cpm. Net cpm values were derived from antiserathat gave the highest binding.

Example 6 Identification of Immunodominant Regions of BoNT/B

This example shows the identification of several immunodominant regionsof human anti-BoNT antibodies within the heavy chain of BoNT/B.

The antigenic regions of BoNT were determined using anti-BoNT antiseraobtained from human, horse and mouse, as shown in Examples 2 through 4.The location of antigenic regions can be narrowed to shorter domains bythe following analysis.

In this analysis, the size of an antigenic site was assigned to be 10-11residues. The H-chain of BoNT/B was therefore broken down into 10antigenic sites. The 10 antigenic sites are defined in Table 6, below.The table gives the approximate locations of only the antigenic regionsthat bind 15,000 cpm of antibody or greater. Although only theimmunodominant regions are shown in Table 3, regions binding loweramounts of antibodies can be of equivalent immunological significance.In sum, this example shows that BoNT/B immunodominant regions having10-11 residues can be determined based on reactivity of anti-BoNTantisera obtained from human, horse and mouse with BoNT/B peptides.

TABLE 6 Major BoNT/B sites that Bind Human anti-BoNT/B Antibodies H_(N)Domain Regions H_(C) Domain Regions Antigenic Amino Acid ResidueAntigenic Amino Acid Residue Regions of SEQ ID NO: 1 Regions of SEQ IDNO: 1 NR1 616-626 CR1 867-877 NR2 735-745 CR2 895-905 NR3 778-789 CR3929-939 CR4 974-984 CR5 1039-1049 CR6 1065-1075 CR7 1269-1281

Example 7 BoNT/B Immunophereses in an Individual

This example illustrates an immunophereses method for treating blood inan individual using an anti-BoNT/B immunoaffinity column.

An individual is connected to an extracorporeal circulation circuitwhere blood is continuously drawn from an antecubital vein via a15-gauge dialysis needle at a flow rate of approximately 50 to 80mL/min. The total volume of blood removed from an individual isapproximately 300 ml. The processed blood is returned back to theindividual. To avoid thrombotic complications, heparin at an input rateof 20 units/min (not to exceed 5000 units per treatment) andanti-coagulant citrate dextrose formula A solution (ACD-A; BaxterHealthcare Corp, Deerfield, Ill.) is administered immediately prior tothe procedure to prevent coagulation. The ratio of citrate to wholeblood was kept at 1:22.

For primary plasma separation, an autopheresis-Ctm Therapeutic PlasmaSystem (TPS) is employed (Baxter Healthcare Corp, Deerfield, Ill.).Plasma is first separated from the cellular components using thePlasmacell-CR, a rotating cylindrical membrane housed in a plasticcasting. The plasma is then directed to a first 150 ml anti-BoNT/Bimmunoaffinity column where anti-BoNT/B antibodies bind to theimmobilized BoNT/B peptides disclosed in the present specification. Theperfusion rate of plasma passing through the column is between 15 to 40ml/min. A continuous flow operation is performed in which the TPS isconnected with an adsorption-desorption-automate (ADA; Baxter HealthcareCorp, Deerfield, Ill.) controlling the flow of plasma and regenerationsolutions. In general, a predetermined amount of plasma is processedthrough the first column and then the flow is directed to a second 150ml anti-BoNT/B immunoaffinity column. While the second column is beingloaded, the first column is regenerated using a suitable low pH buffer.Thus, the columns are alternately loaded and regenerated.

After passage though the anti-BoNT/B immunoaffinity column, the treatedplasma is reconstituted with the cellular components of the blood andreturned back into the individual. The blood can be pre-warmed to bodytemperature before being returned to the individual. This process isrepeated until the desired amount of anti-BoNT/B antibodies iseliminated from the individual's blood.

Example 8 BoNT/B Immunophereses in an Individual

This example demonstrates that an in vitro assay can be used todetermine amounts of blocking or protective antibodies against BoNT/B inhuman serum samples. This example further demonstrates that acombination assay using, for example, two or three synthetic BoNT/Apeptides can be used for sensitive detection of the presence of specificanti-toxin antibodies in, for example, BOTOX® treated patients.

A. Methods for Data Analysis

Mouse protection assay (MPA)-positive cervical dystonia (CD) serumsamples were obtained from Allergan, Parkinson's Disease Center andMovement Disorders Clinic of Baylor College of Medicine, and the ArizonaDystonia Institute. CD patient sera protected against a lethal dose ofBoNT/B in a mouse protection bioassay were screened with 60 synthetictoxin peptides corresponding to the entire H chain of BoNT/B (FIG. 1).Sera from ten individuals who were never treated with BoNT/B was used asa negative control. A 2.5 μg aliquot of each of the 60 syntheticoverlapping peptides was dissolved in 50 μl of 0.01 M phosphate buffer,pH 7.2 containing 0.15 M NaCl (1.0 μg/50 μl of PBS), added to threewells of a flexible polyvinyl chloride 96-well plate. Peptides wereallowed to bind for two hours at 37° C. followed by overnight incubationat 4° C. Plates were washed five times with PBS to remove unboundpeptide and then blocked for one hour at 37° C. with 0.5% bovine serumalbumin in PBS (BSA/PBS). An appropriate volume of each of theMPA-positive CD sera was preincubated with an equal volume of TeNTtoxoid (1 mg/ml) for three hours at 37° C. after which it was diluted to1:500 (vol/vol) with 0.1% BSA/PBS, pipetted (50 μl) into peptide-coatedwells and allowed to react for three hours at 37° C. followed by furtherincubation overnight at 4° C. After washing the wells five times withPBS, 50 μl of prediluted (1:500 vol/vol, in 0.1% BSA/PBS) immunoglobulinfraction of rabbit anti-human IgG (DAKO Corporation; Carpinteria, CAA0424)+IgM (Mu chain; DAKO, A0426) was added and allowed to react at 37°C. for two hours. The wells were washed five times with PBS followed byaddition of 50 μl of ¹²⁵I-Protein A (2×10⁵ CPM in 0.1% BSA/PBS) to eachwell and incubation for two hours at room temperature. Finally, plateswere washed thoroughly to eliminate unbound radioactivity; individualwells were cut out and transferred into separate tubes; and theincorporated radioactivity was counted in a gamma-counter (1277 GammaMaster; LKB, Finland). The results, which were obtained from triplicateanalyses, were expressed as the ratios of mean CPM bound by peptidesover CPM bound by control peptides or bovine serum albumin (BSA).

For determining antibody binding to BoNT/B, triplicate wells were coatedwith the appropriate inactive BoNT/B toxin (0.5 μg/50 μl of PBS). Asimilar procedure was then used to determine the amount of antibodybound by BoNT/B using human MPA-positive CD sera pre-absorbed with TeNT.

B. Mapping of Epitopes Recognized by Antibodies in of MPA-Positive Seraof Cervical Dystonia Patients

The results of mapping by the synthetic H-chain peptides of antibodiesfrom 100 CD patients that were MPA positive was determined. Although thepeptide recognition profiles varied quantitatively and qualitativelyfrom patient serum to patient serum, there were some peptides that wereclearly immunodominant in most patient sera. In order to easily discernthe predominant recognition pattern, we made a pool of equal volumes ofthe 30 CD antisera and analyzed the anti-BoNT/B-antibody binding profileof the serum mixture at a dilution of 1:500 vol/vol. These resultsshowed that high levels of anti-BoNT/B antibodies were bound indecreasing order by peptides C1, C10 and C16, and C14. Medium levels ofanti-BoNT/B-antibody were bound by peptides: N15, N21/N22 overlap,N24/N25 overlap and N29. Peptides N3/N4, N27, C2, C4, C6/C7, C17, C24,C29 and C31 exhibited low anti-BoNT/B-antibody binding. The remainingpeptides showed very low or no detectable anti-BoNT/B-antibody binding.It should also be noted that the antiserum did not bind to unrelated (toBoNT) proteins and peptides.

To determine the frequency of recognition of a given peptide by thepatient sera, results were expressed in percent of antisera which hadanti-BoNT/B-antibody that bound to each of the 60 peptides (FIG. 4).Twenty-eight out of 30 patient pools (93.3%) had anti-BoNT/B-antibodythat bound to peptides C1, C10 or C14 and C16. The two antisera that hadno anti-BoNT/B-antibody-binding to peptide C16 also did not bind to C17.Twenty seven of 30 antisera (90.0%) recognized peptides N22. Two of theantisera that did not recognize peptide N22 also had noanti-BoNT/B-antibody-binding to peptide N21. Of the 30 antisera, 21(70.0%) showed anti-BoNT/B-antibody-binding to peptides N24; 20 (66.7%)had anti-BoNT/B-antibodies that bound to peptide N15, 16 (53.3%)recognized peptide N25 but the overlap N24/N25 had a higher recognitionlevel (21 of 30 or 70.0%); 11 (36.6.3%) recognized the N3/N4 overlap;and 14 (36.6%) bound to peptide N21. Less than half of the patients hadanti-BoNT/B-antibodies against peptides N27, N29, C2, C4, C7, C17, C21,C22, C29, and C31. The remaining peptides either did not bindanti-BoNT/B-antibodies or bound very low levels in 6 or less of thesera. The peptide combinations N24+C1, N22+N24+C1, N24+C1+C10,C10+C14+C16, N22+N24+C1+C10, C1+C10+C14+C16 or N22+N24+C1+C10+C14 wererecognized by all 30 (100%) of the antisera containing neutralizinganti-BoNT/B-antibodies (Table 7).

TABLE 7 Detection of Neutralizing anti-BoNT/B Antibodies in Sera ofMPA-positive CD patients by BoNT/B Peptide Combinations^(a) NumberPercent Detection of of Patients Neutralizing anti-BoNT/B PeptideCombinations Tested Antibodies BoNT/B 30  100% (30/30) N24 + C1 30  100%(30/30) N22 + N24 30 96.7% (29/30) N22 + C1 30 96.7% (29/30) C1 + C10 3096.7% (29/30) C10 + C14 30 96.7% (29/30) C14 + C16 30 96.7% (29/30)N22 + N24 + C1 30  100% (30/30) N24 + C1 + C10 30  100% (30/30) C10 +C14 + C16 30  100% (30/30) C1 + C10 + C14 30 96.7% (29/30) N22 + N24 +C1 + C10 30  100% (30/30) C1 + C10 + C14 + C16 30  100% (30/30) N22 +C1 + C10 + C14 30 96.7% (29/30) N22 + N24 + C1 + C10 + C14 30  100%(30/30) ^(a)The table summarizes the levels of detection by analysis ofblocking Abs binding to various combinations of the six most activepeptides (N22, N24, C1, C10, C14 and C16 (FIG. 7).

1. A BoNT/B peptide having a length of at most 60 amino acids, the BoNT/B peptide comprising an amino acid sequence of amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, or amino acids 1058-1076 of SEQ ID NO:
 7. 2. The BoNT/B peptide according to any one of claim 1, wherein said BoNT/B peptide has a length of at most 45 amino acids.
 3. The BoNT/B peptide according to any one of claim 1, wherein said BoNT/B peptide has a length of at most 30 amino acids.
 4. The BoNT/B peptide according to claim 1, wherein the BoNT/B peptide consists of amino acid sequence of amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, or amino acids 1058-1076 of SEQ ID NO:
 7. 5. A method of determining immunoresistance to botulinum toxin therapy in an individual, the method comprising the steps of: a) combining a BoNT/B peptide and a test sample under conditions suitable for the selective binding of the BoNT/B peptide to an anti-BoNT antibody, the BoNT/B peptide having a length of at least 5 amino acids and at most 60 amino acids and comprising the amino acid sequence of amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of each amino acid sequence thereof, or an immunogenic fragment of each amino acid sequence thereof; and b) detecting the presence of an anti-BoNT antibody-BoNT/B peptide complex, the antibody-peptide complex formed by the selective binding of an anti-BoNT antibody and the BoNT/B peptide; where the presence of the anti-BoNT antibody-BoNT/B peptide complex is indicative of immunoresistance to a BoNT therapy.
 6. The method according to claim 5, wherein the method comprises a further step of correlating the amount of an antibody-peptide complex formed from the test sample relative to the amount of an antibody-peptide complex formed by the BoNT/B peptide combined to a control sample.
 7. The method according to claim 5, wherein the BoNT/B peptide consists of amino acids 736-754 of SEQ ID NO: 1, amino acids 736-754 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 1, amino acids 764-782 of SEQ ID NO: 3, amino acids 764-782 of SEQ ID NO: 5, amino acids 764-782 of SEQ ID NO: 7, amino acids 848-866 of SEQ ID NO: 1, amino acids 848-866 of SEQ ID NO: 3, amino acids 848-866 of SEQ ID NO: 5, amino acids 848-866 of SEQ ID NO: 7, amino acids 974-992 of SEQ ID NO: 1, amino acids 974-992 of SEQ ID NO: 3, amino acids 974-992 of SEQ ID NO: 7, amino acids 1030-1048 of SEQ ID NO: 1, amino acids 1030-1048 of SEQ ID NO: 3, amino acids 1030-1048 of SEQ ID NO: 5, amino acids 1030-1048 of SEQ ID NO: 7, amino acids 1058-1076 of SEQ ID NO: 1, amino acids 1058-1076 of SEQ ID NO: 3, amino acids 1058-1076 of SEQ ID NO: 5, amino acids 1058-1076 of SEQ ID NO: 7, a conservative variant of each amino acid sequence thereof, or an immunogenic fragment of each amino acid sequence thereof.
 8. The method according to claim 5, wherein a portion of the BoNT/B peptide immunoreactive with an anti-BoNT antibody comprises at least six consecutive amino acids of 736-754 of SEQ ID NO: 1, at least six consecutive amino acids of amino acids 736-754 of SEQ ID NO: 5, at least six consecutive amino acids of amino acids 764-782 of SEQ ID NO: 1, at least six consecutive amino acids of amino acids 764-782 of SEQ ID NO: 3, at least six consecutive amino acids of amino acids 764-782 of SEQ ID NO: 5, at least six consecutive amino acids of amino acids 764-782 of SEQ ID NO: 7, at least six consecutive amino acids of amino acids 848-866 of SEQ ID NO: 1, at least six consecutive amino acids of amino acids 848-866 of SEQ ID NO: 3, at least six consecutive amino acids of amino acids 848-866 of SEQ ID NO: 5, at least six consecutive amino acids of amino acids 848-866 of SEQ ID NO: 7, at least six consecutive amino acids of amino acids 974-992 of SEQ ID NO: 1, at least six consecutive amino acids of amino acids 974-992 of SEQ ID NO: 3, at least six consecutive amino acids of amino acids 974-992 of SEQ ID NO: 7, at least six consecutive amino acids of amino acids 1030-1048 of SEQ ID NO: 1, at least six consecutive amino acids of amino acids 1030-1048 of SEQ ID NO: 3, at least six consecutive amino acids of amino acids 1030-1048 of SEQ ID NO: 5, at least six consecutive amino acids of amino acids 1030-1048 of SEQ ID NO: 7, at least six consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 1, at least six consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 3, at least six consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 5, at least six consecutive amino acids of amino acids 1058-1076 of SEQ ID NO: 7
 9. The method according to claim 5, wherein the test sample comprises blood.
 10. The method according to claim 5, wherein the test sample comprises serum.
 11. The method according to claim 10, wherein the test sample comprises an IgG antibody component separated from the serum.
 12. The method according to claim 5, wherein the immunoresistance to botulinum toxin therapy is selected from the group consisting of a BoNT/A immunoresistance, a BoNT/B immunoresistance, a BoNT/C1 immunoresistance, a BoNT/D immunoresistance, a BoNT/E immunoresistance, a BoNT/F immunoresistance and a BoNT/G immunoresistance.
 13. The method according to claim 12, wherein the immunoresistance to botulinum toxin therapy is a BoNT/B immunoresistance.
 14. The method according to claim 5, wherein the individual is a human.
 15. The method according to claim 5, wherein the presence of an anti-BoNT antibody-BoNT/B peptide complex is determined qualitatively.
 16. The method according to claim 5, wherein the presence of an anti-BoNT antibody-BoNT/B peptide complex is determined quantitatively.
 17. The method according to claim 5, wherein the presence of an anti-BoNT antibody-BoNT/B peptide complex is determined using a competitive assay.
 18. The method according to claim 5, wherein the presence of an anti-BoNT antibody-BoNT/B peptide complex is determined using a non-competitive assay.
 19. The method according to claim 5, wherein the presence of an anti-BoNT antibody-BoNT/B peptide complex is determined by an assay format selected from the group consisting of a radioimmunoassay, an enzyme-linked immunosorbent assay, an enzyme immunoassay, a fluorescence immunoassay, and a luminescent immunoassay.
 20. The method according to claim 5, wherein the presence of at least 10% complex formation of an anti-BoNT antibody to a BoNT/B peptide is indicative of a BoNT immunoresistance.
 21. The method according to claim 5, wherein the presence of at most 10% complex formation of an anti-BoNT antibody to a BoNT/B peptide is indicative of a BoNT immunoresistance.
 22. An anti-BoNT immunoapheresis method of treating immunoresistance to a botulinum toxin therapy in an individual, the method comprising the steps of: a) contacting an anti-BoNT antibody containing component from the individual extracorporeally with a BoNT/B peptide immunosorbent under conditions suitable for the selective binding of the BoNT/B peptide to the anti-BoNT antibody, the BoNT/B peptide having a length of at least 5 amino acids and at most 60 amino acids; and b) returning the anti-BoNT antibody depleted component back into the individual.
 23. The immunoapheresis method according to claim 22, wherein the anti-BoNT antibody containing component comprises blood.
 24. The immunoapheresis method according to claim 22, wherein the anti-BoNT antibody containing component comprises serum.
 25. The immunoapheresis method according to claim 24, wherein an IgG antibody component from the serum is contacted a BoNT/B peptide immunosorbent.
 26. The immunoapheresis method according to claim 22, wherein the immunoresistance to botulinum toxin therapy is selected from the group consisting of a BoNT/A immunoresistance, a BoNT/B immunoresistance, a BoNT/C1 immunoresistance, a BoNT/D immunoresistance, a BoNT/E immunoresistance, a BoNT/F immunoresistance and a BoNT/G immunoresistance.
 27. The method according to claim 26, wherein the immunoresistance to botulinum toxin therapy is a BoNT/B immunoresistance.
 28. The immunoapheresis method according to claim 27, wherein the individual is a human.
 29. The immunoapheresis method according to claim 22, wherein the amount of anti-BoNT antibodies removed from an anti-BoNT antibody containing component is at least 10% of the anti-BoNT antibodies from the anti-BoNT antibody containing component.
 30. The immunoapheresis method according to claim 22, wherein the amount of anti-BoNT antibodies removed from an anti-BoNT antibody containing component is at most 10% of the anti-BoNT antibodies from the anti-BoNT antibody containing component. 